Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via...

PLENARY 1 Asia-Pacific Journal of Molecular Medicine 2013, 3 (SUPP 1)

Abstracts for the 1st National Conference for Cancer Research &

5th Regional Conference on Molecular Medicine (RCMM)

8-10th November 2013, The Royale Chulan, Kuala Lumpur

Changing Trends of Chronic Diseases in Malaysia:

Perspective From A Cohort Study

Rahman Jamal

UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia

ABSTRACT

The prevalence of non-communicable diseases (NCDs) is showing an increasing trend in

most developed and developing nations. For diseases such as Type 2 diabetes mellitus and

hypertension, the prevalence is already at a crisis level. Despite intensive efforts and billions

of dollars spent in terms of research, health education and preventive strategies, there is still

no solution in sight as the number of deaths attributable to the NCDs continues to rise. On the

research front, there is still a lot more to be done and arguably the best approach would be

setting up ‘population laboratories’ and studying individuals over time i.e. a cohort design.

The Malaysian Cohort is a one of the latest cohorts amongst the developing nations. But

having a population with 3 major ethnic groups gives the cohort an added advantage in terms

of comparative analysis across ethnic groups. At recruitment written informed consent was

taken and each participant went through a questionnaire-based interview, a biophysical

examination, and gave blood and urine for baseline tests and also storage. The project has

recruited a total 106,527 participants aged 35 years and above from the year 2007 to 2012.

53% were females and 47% males. A total of 71.4% or the participants are from the urban

areas whilst 28.6% are from the rural communities. The prevalence of Type 2 diabetes

mellitus, amongst the Cohort population is 16.6%, hypertension at 46.5%, obesity at 17.7%

and hypercholesterolemia (>5.2 mmol/L) at 75.7%. The percentage of those with impaired

fasting glucose is 10.4%. Clearly the trends are increasing when compared to the data from

the National Health Morbidity Survey by the Ministry of Health Malaysia where in 2006, the

prevalence of diabetes was at 14.6%. Clearly, the lack of physical activity and also poor diet

practices have caused this surge in numbers of those with NCDs. There are already 1131

deaths so far and 226 are due to cancers. The majority of those who died of cancers were

asymptomatic when they were recruited hence giving the opportunity to analyse the blood

specimens for biomarkers for early detection. There is a lot of analysis which can be done on

the baseline data to identify risk factors. The follow-up phase has just started and more than

2500 participants have been revisited so far and re-interviewed, re-measured plus also having

another set of biospecimens taken. An intervention study on those with impaired fasting

glucose is also being planned. In summary, the Malaysian Cohort study confirms the statistics

which we already known but the data and biospecimens will provide endless possibilities in

terms of research especially in terms of looking at gene-environment interaction and also

comparison across ethnic groups. Identification of risk factors, whether lifestyle or genetic in

origin, will be crucial in terms of applying the personalised, preventive and participatory

approaches. Like any cohort study, the value of the Malaysian Cohort will certainly increase

with time.





The Global Cancer Crisis: Needs, Challenges and Opportunities

for Developing Nations

Chia Kee Seng

National University of Singapore, Singapore

ABSTRACT

Across the world, cancer is a major health challenge in urgent need of addressing. The World Health

Organization estimated in 2008 that globally, cancer claims more than 7.6 million deaths annually —

more than HIV/AIDS, tuberculosis and malaria combined. Of these, 70 per cent occurred in low to

middle-income countries. Furthermore, deaths from cancer worldwide are projected to rise to over

13.1 million in 2030, of which 8.9 million are expected to come from the developing world. In and of

itself, cancer prevention does not appear to be an insurmountable challenge. The WHO attributes

approximately 30 percent of cancer deaths to five key modifiable lifestyle-related risk factors: obesity

and overweight, unhealthy dietary habits, a lack of physical activity, smoking, and alcohol use.

However, alongside these challenges, developing contexts struggle with further issues. It has been

estimated that over a third of cancers can be prevented if detected early, but late diagnosis remains a

major issue in developing countries, where about 70 percent of all cancer cases are diagnosed too late.

In addition, cancer survival tends to be poorer in developing countries due to a combination of late

stage cancer at diagnosis and limited access to timely and standard treatment. Much of this can be

attributed to unmet response capacity needs in developing-country settings, spanning the spectrum

from prevention and screening through to diagnosis and treatment. Furthermore, the developing

world is not spared of the global population ageing and rapid urbanization trends, both of which

contribute to rising cancer morbidity and mortality. The recent developments in genomics also present

a challenge and opportunity for cancer control in developing countries. Despite these challenges,

developing countries present great opportunities for cancer prevention and control. These

environments can challenge public health practitioners to look beyond generalized and prescriptive

methods of tackling cancer and instead consider flexible, cost-effective prevention and treatment

alternatives to fit the needs of heterogeneous resource levels, socio-cultural contexts, and economic

settings. This opportunity for innovation and growth in the cancer prevention and treatment – as well

as improvement of the lives of millions across the developing world - should not go unharnessed by

public health.

PLENARY 3

Asia-Pacific Journal of Molecular Medicine 2013, 3 (SUPP 1)




Lifestyle Prevention of Cancer: National Strategies

Lokman Hakim S

Ministry of Health Malaysia

ABSTRACT

Cancer is one of the most devastating diseases, with no single known cause and everyone is at risk. It

causes tremendous suffering and burden to patients, families and societies and is a leading cause of

mortality globally. Incidence of cancer is expected to rise with increasing ageing population,

increasing exposure to carcinogens and growing adoption of unhealthy lifestyles. Major modifiable

cancer lifestyles risk factors include tobacco use, unhealthy diet, physical inactivity and alcohol use.

Tobacco use is the most important risk factor for cancer causing 22% of global cancer deaths and 71%

of global lung cancer deaths. Poor intake of dietary fiber, lack of physical activities and alcohol use

are also associated with increased risks of various cancers. Certain cancers associated with infection

are also related to lifestyle. More than 30% of cancer deaths could be prevented by modifying or

avoiding lifestyle-related risk factors. Cancer share the same risk factors with the other non-

communicable diseases. The National Strategic Plan for Non-Communicable Diseases (NSPNCD)

outlined 7 strategies to address the disease burden of NCDs including cancer. The main strategies can

be broadly grouped into 4 areas namely 1. Community empowerment: transforming community

healthcare behavior, 2. Strengthening inter-sectoral cooperation and collaboration, 3. Enhancing

public health regulation and enforcement, and 4. Enhancing governance and stewardship. It requires

the “whole of government” and “whole-of-society” approach to ensure sustainability of activities and

programs to achieve the desired outcomes.

PLENARY 4





Personalized Medicine: Past, Present and Future

Rodney J, Scott

Discipline of Medical Genetics, School of Biomedical Sciences & Pharmacy, University of Newcastle, Newcastle, NSW, Australia

ABSTRACT

“Personalized medicine” has become a common catch phrase that encompasses many aspects of

medical care. In the traditional sense, we all like to think that we are unique and that anyone involved

in our care is paying special attention to our specific needs – or providing personalized care. This is

not a new phenomenon but our interpretation of it is. The notion that each and everyone of us is

unique has been gaining support ever since the human genome project was completed but even before

that it was becoming apparent that there are individual differences between each and everyone of us.

It is really only over the past 15 or so years that it has become obvious that unique differences within

an individual can be exploited for the benefit of patient care. Personalized medicine has recently taken

some enormous leaps forward as a result of astounding developments in technology. Today it is now

possible to sequence an entire genome within a few days compared to several years just one decade

ago. Commensurate with this quantum leap in ability has been the actual costs of undertaking a

genome sequence. The changing landscape of genomics has resulted in a number of key

developments in our understanding of disease. The first evidence of a change in our understanding of

personalized medicine was the interrogation of locus specific disease once the genes had been

identified. The first genes to be interrogated included TP53, APC, BRCA1, BRCA2 and four DNA

mismatch repair genes. Together, these accounted for a small but significant proportion of patients

who would benefit from individualized care. As information on familial forms of cancer

accumulated there were parallel developments in array technology that culminated in the ability to

screen thousands of markers across the genome, which lead to the introduction of genome wide

association studies (GWAS). GWASs are agnostic approaches to identifying genetic loci associated

with disease and as such they have revolutionized our understanding of disease. More recently,

developments in massively paralleled DNA sequencing have again altered our approach to disease.

Due to the unparalleled changes in DNA sequencing technology it is now conceivable that genetic

information will become available for personal medical care. This will alter healthcare outcomes and

transition health care from a reactive to a predictive profession, tailoring differences in our genomes

to our own individual health care needs.

PLENARY 5





Economics of Cancer Therapy: Can We Afford To Provide Universal

Coverage For Cancer Patients?

Syed Mohamed Aljunid bin Syed Junid

UnitedNations UniversityInternational Institute For Global Health, Malaysia





Central Role of Regulatory RNA in Cancer

John Mattick

Garvan Institute, Australia

Lunch Talk 1





Advancing Clinical Research with Ion Torrent and Ampliseq™ Technology

Michael Richardson

Analisa Resources Sdn Bhd

ABSTRACT

Utilization of genetic information in a clinical setting requires rapid high throughput approaches

compatible with low input amounts of DNA or RNA materials from clinical samples, including FFPE

samples. To meet these challenges, clinical laboratories are transitioning from traditional Sanger

sequencing methods to next-generation sequencing (NGS) technologies. The Ion Torrent

semiconductor sequencing technology directly translates chemical signals into digital information

making it the fastest NGS platform available today. The Ion Ampliseq™ technology enables the rapid

targeted resequencing of hundreds of known mutations or select panels of genes, becoming the most

powerful tool in the determination of sequence variations in specific regions of the genome. This

simple, single day workflow requires very little input amounts of DNA or RNA and is as easy as

setting up a PCR reaction. Ion Ampliseq™ readymade and community panels enable the detection of

cancer causing mutations, inherited diseases and the sequencing of whole exomes. The combination

of Ion Ampliseq™ technology, Ion Torrent sequencers and Ion Reporter analysis software provides a

simple, scalable, rapid and cost effective NGS solution for any healthcare providers.

Lunch Talk 2





Pathway-informed Multi-Omics Analyses: Metabolomics as an

Integral Member of Integrated Biology

Thomas Patrick Hennessy

Agilent Technologies

ABSTRACT

In the last two decades significant progress has been made in the sequencing of the genomes of a

number of different organisms. Simultaneously, large investments have occurred in the development

of high throughput analytical approaches to analyse different cell products, such as those from gene

expression (transcriptomics) as well as proteins (proteomics) and metabolites (metabolomics). These

‘omics approaches, when used in combination with sophisticated bioinformatic tools for data mining

and interpretation, are considered important tools to be applied and utilised to understand the biology

of an organism and its response to environmental stimuli or genetic perturbation. The ability to

measure all elements of a system, such as DNA, mRNA, proteins, metabolites and structural elements

such as the cytoskeleton, cell walls and membranes, and also to determine the relationship of those

elements to one another as part of the system’s response to various stimuli is called Systems Biology.

Metabolomics comprises the combination of high-throughput analytical technologies for the detection

and quantification of metabolites in biological systems with the application of sophisticated

bioinformatic tools for data mining and analysis. In order to analyse the metabolic complement,

different analytical platforms have to be used which results in difficulties integrating data sets derived

from those instruments. In addition, when integrated with data from other ‘omics analysis it is

important to follow certain protocols and procedures to allow for integration. Here we present

workflows integrating comprehensive and multiplatform metabolomics analyses with other ‘omics

data sets allowing for comparative pathway analyses using sophisticated software tools.

Symposium 1 ( i )





Beyond BRCA Mutation in Hereditary Breast Cancer

1,2

Soo-Hwang Teo

1Cancer Research Initiatives Foundation, 2University Malaya Medical Centre

ABSTRACT

It is estimated that BRCA1 and BRCA2 account for approximately 25% of excess familial risk to

breast cancer. In my talk, I will review our molecular understanding of BRCA1 and BRCA2 and the

other genes that contribute to hereditary breast cancer, summarise what we current know about the

risks associated with each gene and provide an update of what we currently know about the relevance

of these genes to the Asian population. This includes rare high penetrance genes such as TP53 and

PTEN, and moderate penetrance genes including ATM, CHEK2 and PALB2. Finally, I will briefly

provide an update on our current understanding of other genes and genetic loci identified through

genome wide association studies and discuss whether these SNP based testing are ready for clinical

practice.

Symposium 1( ii )





Molecular and Genomic Approaches in Understanding the Tumorigenesis of

Oral Cancer and Their Clinical Significance

1, 2

R,B, Zain

1Oral Cancer Research and Coordinating Centre (OCRCC), 2Department of Oro-Maxillofacial Surgical and Medical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia.

ABSTRACT

Part of the challenges in current available prognostic/predictive indicators for the evaluation of

malignancies for the management of cancers lie in the existence of genetic heterogeneity within

tumours.With the development of technologies that allows interrogation of the human genome to

minute details, the changes that occur in cancer can be identified. The possibility of using genomic

profiling studies to subclassify head and neck tumors have been promising but not without barriers.

Amongst the current technologies, identifying copy number alterations using ultra-high resolution

array comparative genomic hybridization (aCGH) has been used to analyse genomic changes in oral

cancers. This presentation will include an overview of (i) our current findings on genomewide

profiling of oral cancer;(ii) a review on copy number alterations (CNAs) in oral cancer and; (iii)an

illustration on the validation processes of candidate genes as potential biomarkers for prognostication

and prediction of clinical outcome in OSCC.

Symposium 2 ( i )





Dealing with Tumor Heterogeneity and Multi-dimensional datasets for

Uncovering Patient-Specific Driver Mutations

Niranjan Nagarajan

Genome Institute of Singapore, Singapore

ABSTRACT

Heterogeneity in cancer both at the tissue level and at the population level continues to pose a

challenge for the interpretation of high-throughput cancer genomics datasets, in stark contrast to the

ease of data generation. In this talk, I will highlight the key issues in this area and how

bioinformaticians are striving to address them. In particular, I will present ideas from the development

of a new somatic variant caller (LoFreq-Somatic) that calls rare variants in heterogeneous tumors with

high sensitivity and specificity. I will also outline a novel approach to integrate diverse –omics

datasets for the prediction of patient specific driver mutations. This approach can be used to combine

large public tumor sequencing datasets (e.g. from TCGA or ICGC) with in-house datasets to interpret

and characterize them.

Symposium 2 ( ii )





Integrating Genomic Data: Towards Identification of Molecular Pathways

Zhibo Gao

Beijing Genome Institute, China

Symposium 3 ( i )





GWAS and NGS in Colorectal Cancer: Risk Management and

Improving Treatment

Rodney J, Scott

Discipline of Medical Genetics, School of Biomedical Sciences & Pharmacy,

University of Newcastle, Newcastle, NSW, Australia

ABSTRACT

Understanding the genetics of colorectal cancer has been advancing ever since the first linkage

analyses were performed on patients diagnosed with disease predispositions such as familial

adenomatous polyposis and Lynch syndrome. Since the introduction of genome wide association

studies aimed at identifying the genetic basis of colorectal cancer many insights into the pathogenesis

of this disease have been forthcoming. Currently, there are at least 25 low penetrance genes

associated with colorectal cancer, all with small effect sizes. On current estimates this accounts for

only ~8% of disease risk, suggesting that a considerably larger number of genetic alterations remain

to be identified. The use of genome wide association studies to identify genetic determinants of

colorectal cancer may be coming to a close as a result of significant gains in our ability to perform

DNA sequencing. Recent studies aimed at sequencing cancer genomes have revealed several salient

points. First, colorectal cancers arising in different sites within the colon and rectum have remarkably

similar mutation profiles; second, there appear to be colorectal specific mutation signatures that can

account for a significant proportion of changes identified within a tumour, which has implications

especially for tumours of unknown origin. Third, the improvement in DNA sequencing technology

(i.e. Next Generation Sequencing or NGS) is revolutionizing our ability to identify persons at risk;f

Forth, NGS is the only approach by which the gut microbiome can be analysed in a time efficient

manner and it is to be expected that significant associations between disease and the microbiome will

be forthcoming. The use of NGS is also being used to enhance the use of targeted therapies as it is an

ideal platform by which to identify somatic changes in panels genes. It is to be expected that further

evidence of the value of NGS will be forthcoming in the near future when it is aimed at individual

patients not only to identify somatic changes associated with disease but also provide

pharmacogenetic information about adverse drug reactions.

Symposium 3 ( ii )





GWAS in Breast Cancer: Key Lessons Learned

Chia Kee Seng

National University of Singapore, Singapore

ABSTRACT

Genome-wide association studies (GWAS) examine common genetic variants in different individuals

in order to identify associations between variants and major diseases. One of the diseases that has

arguably benefited most from GWAS developments and discoveries is breast cancer. The GWAS has

yet to convincingly demonstrate much public health and clinical usefulness in helping practitioners

better understand the risk of breast cancer in individuals and populations. It has perhaps provided

better insight into the mechanisms and pathways of carcinogenesis. The GWAS approach has

however, highlighted the great value of the hypothesis-free approach in research and in large-scale

collaborative studies between and among multiple, diverse consortia to identify novel breast cancer

risk loci. Comparisons of multiple cohorts across the world with different genetic profiles have helped

show which risk loci apply across different populations and which are population-specific.

Symposium 4 ( i )





Genes and Obesity

1,2Wan Zurinah Wan Ngah

1UKM Medical Molecular Biology Institute, Kuala Lumpur, 2Department of Biochemistry, Faculty of Medicine, Jalan Raja Muda Abdul

Aziz, 50300, Kuala Lumpur, Malaysia

Symposium 4 ( ii ) Asia-Pacific Journal of Molecular Medicine 2013, 3 (SUPP 1)




Artheroprotective Genes: Do They Exist?

Roshni R Singaraja

Translational Laboratory in Genetic Medicine, A-Star, Singapore

Symposium 5 ( i )





Pharmacogenomics and Ethnicity: Implications for the Treatment of Cancer and

Other Diseases in Global Populations

Liam R. Brunham

Translational Laboratory in Genetic Medicine, A-Star, Singapore

ABSTRACT

Many drugs exhibit marked differences in efficacy and risk of adverse drug reactions between

different ethnic populations. One source of this variability is the difference in frequency of alleles that

modulate drug response. We have investigated the allelic diversity of important drug–

biotransformation and drug–response alleles in South East Asian populations compared to Europeans.

We also compared rates of reported ADRs in these Asian populations to determine if the allelic

differentiation corresponded to an excess of the associated ADR, using ADR reporting data from the

Singapore Health Sciences Authority. Our results point to substantial diversity at specific drug

response loci that may contribute to observed inter-population differences in drug response.

Identification of specific genetic variants that predispose to drug response phenotypes across different

populations can elucidate biological mechanisms for drug response and allow identification of

patients at highest risk for therapeutic failure or drug toxicity.

Symposium 5 ( ii )





Personalised and Precision Medicine Approach in Childhood Leukaemias

Hany Ariffin

University of Malaya

ABSTRACT

Leukemia is the commonest paediatric cancer, afflicting 1:25,000 persons aged less than 15 years

annually. For many decades, leukemia was a ‘death sentence’ but now more than 80% of children

diagnosed with acute lymphoblastic leukemia (ALL) in developed countries are long-term survivors.

This success can be attributed to a combination of factors - the most important being the recognition

that ALL is a heterogenous disease requiring stratified and personalized therapy. With contemporary

chemotherapy, the prognostic strength of traditional clinical presenting features of ALL e.g

immunophenotype and presenting leucocyte count are diminishing, whereas established and newly

discovered genetic and biologic factors are becoming increasingly more important. Early response to

therapy is another strong determinant of disease relapse. Studies of treatment response measured by

more sensitive and objective methods that can detect leukemia cells undetectable by morphology (ie,

minimal residual disease, MRD) have provided a rationale for the incorporation of MRD testing in

risk assignment strategies. In this lecture, the Malaysia-Singapore (Ma-Spore) ALL 2003 multicenter

study will be discussed. This treatment protocol was designed with the premise that combining

clinical and genetic presenting features with molecular assessment of MRD to assess response would

allow precise, personalised therapy for ALL to be delivered with adequate therapeutic intensity yet

with minimal toxicity.

Symposium 5 ( iii )





Mutation Analysis in Gliomas

Roslan Harun

UKM Medical Molecular Biology Institute, Malaysia

ABSTRACT

Gliomas account for 80 % of all primary malignant brain tumors. The most common and aggressive

subtype is glioblastoma (GBM) that carries a poor prognosis with median survival of only 15 months.

The Cancer Genome Atlas (TCGA) Research Network reported a comprehensive analysis of

molecular characteristics of glioblastomas. The TCGA analysis demonstrated that glioblastomas

frequently acquire gains of chromosomes 7 and 19, losses of chromosomes 10 and 13, amplifications

ofEGFR, CDK4, PDGFRA and MDM2, CDKN2A/B deletion, and somatic mutations in TP53, PTEN,

NF1, EGFR, ERBB2, RB1 and PIK3R1. Three critical signalling pathways with frequent genetic

alterations are identified in GBM i.e. RTK/RAS/PI(3)K, p53 and RB signalling pathways. However,

the prevalences of IDH1 and IDH2mutations arehigh in low grade gliomas but low in glioblastomas.

Genome wide association studies (GWAS) offer unbiased interrogations of the genome. Recent

GWAS have identified 7 common susceptibility loci at 5p15.33 (TERT), 8q24.21 (CCDC26), 9p21.3

(CDKN2A-CDKN2B), 20q13.33 (RTEL1), 11q23.3 (PHLDB1), and two independent signals at

7p11.2 (EGFR). Risk alleles in TERT and RTEL1 predispose to an aggressive pathway, whereas

CCDC26 and PHLDB1 risk alleles predispose to lower grade disease. With the next-generation

sequencing, targetedsequencing of specific genes in gliomas can be performed to detect mutations in a

powerful, rapid and scalable manner. The tumour resequencing helps to identify mutations that are

likely to contribute to tumourigenesis (“driver” mutations) ortumour proliferation (“passenger”

mutations). The functional and associations studies may provide novel insights into the biological

mechanisms underlying glioma formation and development.

Symposium 6 ( i )





Point of Care Testing: The Future is Here and Arriving

Avijit Roy

RVR Diagnostics Sdn Bhd

ABSTRACT

Point of Care Tests (POCT) continue to evolve and integrate new science and technology. Yet for all

the hype and promise many such innovations do not become reality in health care settings. By far the

most important driver is reliability and meaningful results in the real world, ie providing tests result

accuracy that are comparable to traditional laboratory tests. The secondary fundamentals that

determine if such tests succeed are speed, cost and ease of use. By incorporating these fundamentals,

immunochromatographic flow devices have moved to a new level of performance. They continue to

evolve and could represent a major paradigm change in how we diagnose and manage disease. In

particular the impact of HIV testing has been substantial. The possibility to adapt this technology to

control dengue fever will also be explored. Immunochromatographic devices have made a

tremendous impact on society and will continue to grow in importance.

Symposium 6 ( ii )





Development of Therapeutic Agents For The Treatment of

Nasopharyngeal Carcinoma

Alan Khoo Soo Beng

Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia

ABSTRACT

Nasopharyngeal carcinoma is a major cancer in Malaysia. Development of agents for therapy is

important to offer alternatives for cases of treatment resistant nasopharyngeal carcinoma (NPC). Our

laboratory has been studying the effects of inhibition of the p53-MDM2 interaction on NPC cells. We

found that nutlin-3 activated the p53 pathway and sensitized NPC cells to the cytotoxic effects of

cisplatin in a p53 dependent manner. We have also been testing the effects of several metal

coordinated ternary complexes on NPC cells. We found a set of copper complexes which displayed

cytotoxic effects against NPC cells, but were less toxic against nasopharyngeal epithelial cells at

similar doses. To assess the anti-cancer activity of promising therapeutic agents for NPC, we sought

to establish primary xenografts of NPC in immunodeficient NOD.Cg-Prkdcscid

Il2rgtm1Wjl

/SzJ mice,

via the subcutaneous and subrenal capsule approach. We have successfully established an EBV

negative primary xenograft from a patient with recurrent NPC and an EBV positive primary xenograft

from a patient with primary NPC. The xenografts are being characterized for further studies.

Symposium 6 ( iii )





Mining the Human Gastric Fluid Proteome for Cancer Biomarkers:

Challenges and Opportunities

Mac Ho Meng Fatt

National Cancer Centre, National University of Singapore, Singapore

ABSTRACT

Gastric cancer (GC) is a significant public health burden, being the second leading cause of cancer-

related deaths worldwide. Its lethality is mainly due to the lack of reliable and clinically acceptable

techniques for diagnosing early-stage GC, for which 5-year survival rates are >90%. Hence, early

detection can significantly reduce mortality and improve outcomes for GC patients. However, there

are currently no biomarkers of acceptable sensitivity and specificity for GC. We have identified

fasting gastric fluid (or gastric juice) to be an excellent source for seeking sensitive biomarkers for

GC because of its anatomic proximity to the site of disease and it is composed of a mixture of secreted

soluble and exfoliated cellular proteins from the gastric mucosa. Importantly, proteins secreted by

tumor cells will be at higher concentration in gastric fluid compared to systemic body fluids (e.g.

blood). We believe the composition and concentration of proteins in gastric fluid reflect the functional

state of the stomach. However, gastric fluid is a complex biological fluid with unique intrinsic

properties. Furthermore, its composition is dynamic, being influenced by physiological,

pharmacological, and pathological factors. In contrast to other body fluids, very little is known about

the gastric fluid proteome. In this presentation, I will describe our findings of the human gastric fluid

proteome and highlight its normal biological variations, which have a profound impact on biomarker

discovery studies. I will discuss the challenges and prospects of biomarker discovery in gastric fluid.

Symposium 7 ( i )





Next Generation Sequencer (NGS) for Single Cell Sequencing

Zhibo Gao

Beijing Genome Institute, China

ABSTRACT

Over the past decades, the whole cancer genome sequencing seems to be impossible. As the next

generation sequencing (NGS) develops, the landscape of human cancer genomes is published

progressively. For most of cancer types, this landscape consists of fewer “mountain” (genes mutated

in high percentage) and a larger number of “hills” (gene altered less frequently). Many “mountain”

genes are reported recently, but “hills” genes hunting still remained to be a challenge. In addition,

after driver mutations of one individual are available, there is another problem on how to clarify their

relationship. These relationships determined the underlying different sub-clones, which have a clue on

cancer evolution. Several published paper based on NGS emerged to provide limited solutions with

multi-sampling. Single-cell sequencing is the ultimate resolution of the multi-sampling approach, and

acts as a potential useful tool to resolve this issue. Therefore, our job is divided into two parts. First,

we sequence more cancers by the whole genome/exome sequencing to identify more driver mutations.

Second, we continuously develop single-cell sequencing to give a comprehensively evolutionary

process of a single tumor, and better knowledge of this process renders more accurate therapy against

cancer and makes personalized medicine possible.

Symposium 7 ( ii )





Proteomics for Biomarker Discovery

Saiful Anuar Karsani

Institute of Biological Sciences, Faculty of Science, University of Malaya and University of Malaya Centre for Proteomics Research (UMCPR), Kuala Lumpur, Malaysia

ABSTRACT

Proteomics is an emerging area of research of the “post-genomic era” and describes the global

analysis of gene expression at the protein level in a multitude of samples. A battery of techniques is

used to resolve (2D electrophoresis, liquid chromatography, protein chips etc.), identify (by peptide

sequencing, mass spectrometry, immunoblotting etc.), quantitate and characterize proteins, as well as

to store and interlink protein and DNA sequence and mapping information from the genome projects.

The demands of proteomics projects have lead to the rapid development of various technologies,

particularly mass spectrometry, which allowed high-throughput identification of proteins in complex

mixtures. Although the mass spectrometry is central in almost all proteomics based research, its

ability to study and characterize biological samples is limited by the sample type, preparation and

analysis tools used. Here we explore the various proteomics approaches and their use in biomarker

discovery. A selection of basic and advanced proteomics techniques, and its promise for cancer

biomarker discovery are discussed.

Symposium 7 ( iii )





In Vitro and In Vivo Models for Cancer Research

Cheong Sok Ching

Oral Cancer Research Team, Cancer Research Initiatives Foundation (CARIF), 12A, Jalan TP5, Taman Perindustrian UEP, 47600 Subang

Jaya, Selangor Darul Ehsan, Malaysia and Oro-Maxillofacial and Medical Sciences Department, Faculty of Dentistry, University of

Malaya, 50603 Kuala Lumpur

ABSTRACT

In vitro and in vivo experimental models are essential tools that facilitate our basic

understanding of disease progression and respond to treatment interventions. In vitro models

can be manipulated with ease to study gene function in the identification of essential genes in

cancer initiation and development. Cultured cancer cells can exhibit distinct properties from

their naturally growing counterparts, however, analysis of large panels of cell lines show that

cell lines do recapitulate the heterogeneity and complexity of cancers and therefore, remain as

important model systems for evaluating candidate anticancer agents. Although cancer cell

lines have become invaluable for the studies of genetics, pharmacology, and in vitro cell

biology,they fail represent the tumour in totality, and does not consider factors in the tumour

microenvironment that underlie tumor behavior. For this, several animal models have been

established for the in vivo study of carcinogenesis, tumorigenesis and cancer

treatment.Drawing from the experience we have in establishing models for oral squamous

cell carcinoma, I will discuss the various model systems available for cancer research, the

limitations that are inherent to some of these models and most importantly how to we can use

them to improve our understanding of cancer development.

Symposium 8 ( i )





Oxidative Stress and Ageing

1,2

Wan Zurinah Wan Ngah

1UKM Medical Molecular Biology Institute, Kuala Lumpur, 2Department of Biochemistry, Faculty of Medicine, Jalan Raja Muda Abdul

Aziz, 50300, Kuala Lumpur, Malaysia

ABSTRACT

Lifestyle has been suggested to influence the ageing process through affecting common mechanisms

involving oxidative stress and inflammation, thus increasing risk to degenerative diseases. However,

how oxidative stress, singly or together with inflammation contributes to the ageing process and

whether they are causal or as a result of effect of ageing is still uncertain. In vitro and animal models

have provided some insight to the processes involved as variations exist in different populations. A

study conducted in Malaysian men revealed that several parameters such as age and ethnicity affected

DNA damage, plasma oxidation-reduction potential (ORP), protein carbonyl and advanced glycation

end products (AGE). Results from 729 Malay and Chinese men aged 20-80 years showed no

differences (p>0.05) between Malay and Chinese men for DNA damage while protein carbonyl was

higher in Chinese men (p<0.05). Plasma ORP and AGE products were higher in Malay men compared

to Chinese men (p<0.05). A bivariate correlation between age with ORP, DNA damage, protein

carbonyl and AGE for both Malay and Chinese was positive with strong association among them

(p<0.01). Intervention by fasting caloric restriction resulted in a reduction in DNA damage, plasma

ORP, protein carbonyl and AGE after 12 weeks (p<0.05). In attempts to understand the ageing

process in relation to gene expression, peripheral blood mononuclear cells (PBMCs) form

octo/nonagenarians (80-99 years old) and their offspring (50.2 ± 2.0 years old) were studied. Genome

wide microarray analysis revealed 477 transcripts were age-induced and 335 transcripts were age-

repressed in octo/nonagenarians compared to offspring. Interestingly, changes in gene expression

were associated with increased capacity for apoptosis (BAK1), cell cycle regulation (CDKN1B),

metabolic process (LRPAP1), insulin action (IGF2R) and increased immune and inflammatory

response (IL27RA), whereas response to stress (HSPA8), damage stimulus (XRCC6) and chromatin

remodelling (TINF2) pathways were down-regulated in octo/nonagenarians where genes affected

were involved in systemic telomere maintenance, metabolic, cell signalling and redox regulation.

Intervention by supplementing with antioxidants such as vitamin E tocotrienol resulted in reduction in

DNA damage, improvements in oxidative stress markers and inflammatory markers at RNA and

protein expression levels. In conclusion, oxidative stress related mechanisms occur with the ageing

process suggesting avenues for modulation by altering the redox status.

Symposium 8 ( ii )





Cellular Therapies: Regulatory Issues/Application/Regenerative

Medicine/Cartilage Injuries

Tunku Kamarul Zaman Tunku Zainol Abidin

Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya

Symposium 8 ( iii )





Modulators of Neurodegeneration


Musalmah Mazlan

Medical Faculty, Universiti Teknologi MARA, Sg Buloh, 47000 Selangor, Malaysia

ABSTRACT

Neurodegeneration in brain aging has been suggested to be due to accumulation of free radicals. In

vitro studies have shown that oxidative free radicals can induce neuronal and astrocytes apoptosis.

Thus antioxidants may prove beneficial for preventing neurodegeneration. Our studies using neuronal

cells have shown that antioxidants such as vitamin E and extracts of centella asiatica, piper betel,

momordica charantia, and nigella sativa protected neurons against oxidative stress induced cell

death. In vivo studies in animal models have shown that antioxidant such as vitamin E in the form of

tocotrienol rich fraction (TRF) improved circulating and brain antioxidant status. This improvement

correlates with improvement in cognitive functions as tested using the open field and Morris Water

Maze tests. Molecular studies revealed that tocotrienol and centella asiatica modulate

neurodegeneration by influencing the apoptotic and neuroinflammatory pathways.

Symposium 9 ( i ) Asia-Pacific Journal of Molecular Medicine 2013, 3 (SUPP 1)





Non-coding RNA

John Mattick

Garvan Institute, Australia

Symposium 9 ( ii )





Uncovering Metastatic Cancer through MicroRNA

Norfilza Mohd Mokhtar

UKM Medical Molecular Biology Institute & Department of Physiology, Universiti Kebangsaan Malaysia

ABSTRACT

The tendency of cancer cells to escape from its primary site to the new site to reside is a process

called as metastasis. Most cancer deaths are the result of metastasis sites rather than the primary

lesions. Therefore the emergence of microRNA inhibitors to regulate their targeted genes in order to

stop the process signify promising new targets in cancer treatment. Despite its small size and not

coding or translated to protein, its exists in human cells has a considerable role. The significance of

microRNA in diseases can be witnessed in the recent years with the expanding of the total number of

microRNA in the database, from a few hundreds to a few thousands. The nature of its short sequence

making it easier to target the 3’-untranslated region of genes and interfere with their post-

transcriptional regulations. A specialised group of microRNA called metastamir has been studied in

many cancers covered from the early detection, monitoring, predicting survival and prognosis and

finally targeted treatment. Our current study aimed to identify differentially expressed miRNA with

the mRNA of targeted genes in three different solid tissues namely colorectal cancer, serous ovarian

cancer and prostate cancer. By genome wide expression array and microRNA microarray, we could

identify a list of microRNA interacted with mRNA of the targeted genes. Validation of these

signatures have confirmed the microarray data. We postulate the down-regulation of targeted genes

was most likely due to the post-transcriptional modification by microRNAs. These targeted genes will

be up–regulated upon transfection with the application of specific microRNA inhibitor into in vitro

model of cancer.

Symposium 10 ( i )





The Dark Matter in Your Body: How Human Microbiome Could Change

the Practice of Medicine

Kok-Gan Chan

Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala

Lumpur, Malaysia.

ABSTRACT

Often known as the “another” genome of human being, the microbiome, once considered unknown

due to culturability of the microorganisms present in the human body, is now feasible by coupling

metagenome and the next generation sequencing. Looking at the human body as an ecosystem that

contain trillions of microbial species among themselves and the host, the human health will depend

largely on the very fine balance of the microorganisms population in the microbiome. In evolution,

this fine balance will strike to protect the host and the microbes, in exchange of food and shelter for

the microbes that thrive in and on the human body, and hence protection accorded to the host. But in

the time of turmoil, this fine balance goes off the tangent and the microbiome may misbehave in a

way that causes illness. In this paper, I will discuss illnesses such as diabetes, gastrointestinal

diseases, obesity, asthma, dermatologic diseases, heart disease, urogenital diseases and multiple

sclerosis, oral diseases to neurological conditions such as autism, where the microbiome seems to play

a pivotal role.

Symposium 10 ( ii )





Dengue Genome: Implication for Vaccine Development

Sazaly Abu Bakar

Department of Medical Microbiology, Faculty of Medicine, University of Malaya

YIA-MM1





Tualang Honey Improves Human Corneal Epithelial Progenitor Cell Migration

and Cellular Resistance to Oxidative Stress In Vitro

1Jun-Jie Tan,

1Siti Maisura Azmi,

2Yoke-Keong Yong,

1Hong-Leong Cheah,

1Vuanghao Lim,

1Doblin Sandai,

1,3Bakiah Shaharuddin

1 Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Kepala Batas, Penang, Malaysia, 2 Department of Human Anatomy, Faculty of Medicine and Health Sciences, 43400 Serdang, Selangor Darul Ehsan, Malaysia, 3Institute of Genetic Medicine,

Newcastle University, International Centre for Life,Newcastle Upon Tyne NE1 3BZ, United Kingdom

ABSTRACT

Stem cells with enhanced function and resistance to oxidative stress after in vitro expansion have

shown to improve engraftment and regenerative capacity. Such cells can be generated by

preconditioning them with exposure to an antioxidant. Here we tested the effects of Tualang honeyon

human corneal epithelial progenitor (HCEP) cells in culture. Cellular toxicity, gene expression,

migration and cellular resistance to oxidative stress in response to Tualang honey were evaluated.

Results immunofluorescence staining showed that HCEP cells were holoclonal and expressed

epithelial stem cell marker p63 without corneal cytokeratin 3. The cell viability remained unchanged

after being cultured with 0.004, 0.04 and 0.4% Tualang honey in the medium, but it was significantly

reduced to 63.1±2.1% when the concentration was increased to 3.33%. Cell migration, measured by

gap area occupied by the cells after 48 hours treatment, improved significantly after cultured with

Tualang honey at 0.04% ( p<0.05) and 0.4% (p<0.01). Our data also confirmed that Tualang honey

possessed hydrogen peroxide scavenging capability, albeit trace level of hydrogen peroxide was

detected in Tualang honey in its native form. Interestingly, HCEP cells treated with 0.4% Tualang

honey for 48 hours showed significantly lower dead cells (15.3±0.4%) than the untreated group

(20.5±0.9%, p<0.01) following oxidative insults induced by 50 µM hydrogen peroxide after 24 hours.

Gas chromatography-mass spectrometry (GCMS) analysis revealed that the major constituent in

Tualang honey was 5-hydroxymethyl-2-furancarboxaldehyde, and several other known antioxidant

phytochemicals were also present but at lower levels. This study suggests that Tualang honey possess

antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in

HCEP cells in vitro in a dose dependent manner, but the benefit is offset by its cytotoxic effect at high

concentrations.

YIA-MM2





Inositol 1,4,5-Trisphosphate Receptor 1 in the Rat Midbrain and Brainstem:

Focus on Dopaminergic Cell Populations

1,2

HA Damanhuri, 1NN Kumar and

1AK Goodchild

1The Australian School of Advanced Medicine, Macquarie University, NSW Australia 2Biochemistry Department, Faculty of Medicine,

Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia

ABSTRACT

Inositol triphosphate (IP3) activates IP3 receptors (IP3Rs) on the endoplasmic reticulum to release

calcium into the cytoplasm. IP3Rs play diverse roles in neurons including regulating burst firing,

somatodendritic release of neurotransmitter and determining excitability. The broad descriptions of

the three receptors, IP3R1, IP3R2 and IP3R3, in the central nervous system, suggest they are

differentially distributed however no detailed description of their distribution within the

brainstem/midbrain is available. We have identified the distribution of the most widely abundant

IP3R1, focussing on catecholaminergic cell groups. The distribution of mRNA was determined and

quantified using in situ hybridisation in combination with immunocytochemistry. IP3R1 protein was

detected using western blotting and immunocytochemistry. Our findings demonstrate that IP3R1

mRNA is highly restricted to discrete nuclei in the brainstem and midbrain and its presence closely

reflects IP3R1 protein expression. Abundant IP3R1 mRNA and protein are present in the ventral

midbrain regions that contain catecholaminergic cell groups A8-10 but they are not present in

brainstem catecholaminergic cell groups (A1-7). As Gq coupled receptors are expressed in neurons

within A1-7, IP3 must act via IP3R2 or IP3R3. Quantitative analysis in midbrain dopaminergic cell

groups (A8-10) shows that 25±0.9% of VTA whereas 59±2.4% of SNr neurons expressed IP33R1

mRNA. IP3R1 mRNA was also present in non-dopaminergic neurons in ventral midbrain. Thus these

data demonstrate that IP3R1 is localised in distinct subpopulations of midbrain dopaminergic cells.

Whether this is related to neuronal firing characteristics, other functional properties or neurochemistry

of these cells remains to be determined.

YIA-MM3





α-Factor Analogues Stimulate Pheromone Mating Pathway in S. cerevisiae

1 K Tajul Arifin,

2M Ortmayer,

2S Checkley,

2M Walsh and

3J McCarthy

1 Department of Biochemistry, Universiti Kebangsaan Malaysia, 2 Manchester Interdisciplinary Biocentre, Manchester UK, 3 School of Life

Sciences, The University of Warwick, Coventry UK.

ABSTRACT

The pheromone mating pathway in baker’s yeast Saccharomyces cerevisiae enables this

organism to initiate a developmental response upon detection of a mating pheromone.

Stimulation of the Ste2 receptor by an external trigger molecule (α-factor) in one of the

haploid mating cells of yeast MATa, results in the transcriptional induction of a subset of

yeast genes that are involved in mating. The aim of this study was to convert this system into

a readily measurable response, to function as a biosensor. A successful improved system

should be able to detect other peptides, such as peptide markers in diseases. An in-vivo

luciferase activity assay utilizing a PFUS1:LUC construct, was developed to report the

activation of the pathway. Deletions of both FAR1 and SST2 genes in the experimental strain

were proven to provide an increased signal to noise ratio. As an attempt to decouple Ste2 and

its ligand the α-factor, three α-factor analogues; N3G, E7 and C3G, were synthesized. The

binding activities of the three analogues were assayed by the developed in-vivo luciferase

activity assay. The analogues were found to stimulate the mating pathway in such order:

N3G, E7 and C3G. The result indicated the possibilities of the Ste2 to bind to other novel

peptides. This study not only provided more insight to the physical interaction between Ste2

and novel peptides, but also the possibility of uncoupling a pathway in an already developed

system.

YIA-MM4





B-myb/Dream Complex is Not Critical in CaSki Cell to Regulate the

G2/M Genes

1,2,3

N. Nor Rashid, 1,2

R. Yusof, 3R.J. Watson

1Department of Molecular Medicine, Faculty of Medicine: 2Drug Design and Development Research Group University of Malaya, Kuala

Lumpur, Malaysia: 3Department of Virology, Faculty of Medicine, Imperial College London, London, UK

ABSTRACT

B-myb is a transcription factor that plays role in regulating gene expression and is implicated in

controlling carcinogenesis and cellular senescence. Previous studies have shown that the B-

myb/DREAM complex is required for transit through mitosis in embryonal stem cells which lack

pocket protein/DREAM complex. They also had found a pronounced defect in completing mitosis by

targeting one of the DREAM complex members (Lin-54). Therefore with this understanding, we

wished to investigate whether B-myb/DREAM has a similar function in HPV16-transformed cells,

which also deficient in pocket protein/DREAM complex. This question is significant, because our

previous results showed increased in B-myb/DREAM expression in CaSki and SiHa cells, at least

when compared to the T98G cell control, suggesting that disruption of the pocket protein/DREAM

complex by 16E7 promotes the formation of the transcriptionally active B-myb/DREAM complex,

which could then have a positive impact on cell cycling. Firstly, we depleted the Lin-54 gene in

T98G, SiHa and CaSki cells followed by co-immunoprecipitation with three of the DREAM complex

constituents (Lin-9, Lin-54 and B-myb). Western blot was carried out and probed against B-myb, as

B-myb is highly expressed in CaSki cell. Subsequently, FACS analysis was performed to investigate

the cell cycle progression using the propidium iodide staining method. To investigate how critical the

B-myb/DREAM complex function is in CaSki cell, we further determined the effect of Lin-54

knockdown on mRNA levels of certain S/G2 genes, such as cyclin B, anrora kinase A and Polo-like

kinase 1. We showed that the expression level of these genes was marginally increased in CaSki cell

when compared to the control shRNA transduced cells. These results suggest that B-myb/DREAM

complex is not critical in the cell cycle progression of CaSki cells.

YIA-MM5





Association Analysis of Insulin (INS) and Insulin Receptor (INSR)

Gene Polymorphisms With Obesity in Malay Children

1Christinal Teh Pey Wen,

2Chong Pei Nee,

1Nurul Adibah Nizam,

2Poh Bee Koon,

1Rahman Jamal,

1Wan

Zurinah Wan Ngah

1UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 2Nutritional Sciences Programme, School of

Healthcare Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur

ABSTRACT

Childhood obesity is rapidly emerging as a global epidemic, and the search for genes linked to

obesity-related phenotypes is on-going. INSR gene plays an important role in energy regulation and

studies have shown that the INS gene polymorphisms are associated with obesity-related traits in

Caucasians. There have been no similar studies in the Malay population. Therefore, the aim of this

study was to investigate the association between polymorphisms of the INS gene (rs689) and INSR

gene (rs3745551) with obesity and metabolic phenotypes in Malay children. A total of 528 normal

weight and 557 overweight and obese primary school children aged 6-12 years old were genotyped

using the semi-automated Sequenom iPLEX® Gold assay. Body weight and height were measured,

and body mass index (BMI) was calculated. Waist and hip circumferences were also measured. Body

composition was measured by bioelectrical impedance technique. Blood samples were taken and

analysed for lipid profile and fasting glucose. The genotype distributions of rs689 (T/T: 0.6%, A/T:

15.9% and A/A: 83.5%) and rs3745551 (G/G: 5.4%, A/G: 37.8% and A/A: 56.8%) were in Hardy

Weinberg equilibrium. The minor allele frequency of the T-allele for rs689 and G-allele for

rs3745551 were 8.6% and 24.3%, respectively. Children carrying risk allele of variants of INS gene

had significantly increased risk of obesity (sex- and age-adjusted OR=1.580; 95% CI: 1.134-2.201).

Besides that, polymorphisms of INS gene (rs689) were also found to be associated with increased

body fat percentage [β(95%CI)= 1.646 (0.264-3.029)] and waist circumference [β(95%CI)= 2.000

(0.011-3.989)]. However, the INS gene polymorphisms showed no significant association with

metabolic phenotypes. There is no significant association of INSR gene polymorphisms with obesity-

related and metabolic traits. In conclusion, polymorphisms of the INS gene, but not INSR gene, were

associated with obesity-related traits in the Malay childhood population.

YIA-MM6





Investigating GDF5-Induced Tenogenesis in Bone Marrow Stromal Cells for

the Potential Improvement of Tendon Repair

1SL Tan,

2M Roebuck,

2SP Frostick,

3L Selvaratnam,

1TS Ahmad and

1T Kamarul

1Tissue Engineering Group (TEG), National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), Department of

Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Pantai Valley, Kuala Lumpur 50603, Malaysia, 2Musculoskeletal Science

Research Group, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Daulby Street, Liverpool, L69 3GA, UK, 3School of Medicine & Health Sciences, Monash University, Sunway Campus, Selangor, Malaysia.

ABSTRACT

Growth Differentiation Factor 5 (GDF5) is a tenogenic differentiation inducer in bone marrow stromal

cells (MSCs). The aims of this study were to investigate the effect of GDF5 on human MSCs

(hMSCs) tenogenic differentiation by evaluating the global gene expression profiles as well as

molecular imaging analysis of candidate tenogenic markers and cytoskeleton reorganization. In this

study, hMSCs were isolated and characterized according to ISCT criteria. Then, the cells were treated

with 100 ng/ml of GDF5 to induce tenogenic differentiation. The GDF5-induced hMSCs (at day 4 and

day 10), untreated hMSCs (negative control) and human tenocytes isolated from hamstring tendons

(positive control) were analyzed (n=6) using Human Gene 1.0 ST array (HuGene). The results

showed a significant difference in 954 genes in GDF5-induced hMSCs and tenocytes compared to

control hMSCs. The differentially expressed genes were involved in specific pathways (i.e.

cytoskeleton remodeling, cell adhesion and extracellular matrix related genes) that are closely related

to the native behavior of tenocyte in vivo. Confocal laser scanning microscopy (CLSM) imaging

analysis revealed that the GDF5-induced hMSC expressed higher amount of candidate tenogenic

marker protein. Further, the CLSM analysis also demonstrated that day-4 GDF5-induced hMSCs

possessed more stress fiber arrays which localised primarily next to the cell attachment site.

Following extended GDF5 induction (day 10), MSCs displayed long, thin stress fibres, similar to that

of tenocytes. These observations suggested that GDF5-induced a reorganization of actin structures

which involved F-actin polymerization and restructuring of cortical actin elements to newly formed

stress fibres. The results of atomic force microscopy (AFM) live cells imaging shadowed those

images obtained by CLSM, where stress fibers-like structure can be visualized in the day-10 GDF5-

induced hMSCs. In conclusion, GDF5-induced hMSCs showed a directed differentiation of hMSCs

into tenocyte-like cells, which has a great potential in functional tendon tissue engineering

applications.

YIA-CA1





Gamma-tocotrienol Mimics BH3-only Proteins to Kill Neuroblastoma Cells.

1,Jen-Kit Tan,

2Sue-Mian Then,

3Musalmah Mazlan,

1A Rahman A Jamal,


1 UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia Medical Center (UKMMC), Kuala Lumpur, Malaysia. 2

Faculty of Medicine and Health Sciences, The University of Nottingham Malaysia Campus (UNMC), Semenyih, Selangor, Malaysia. 3 Institute for Medical Molecular Biotechnology (IMMB), Universiti Teknologi MARA (UiTM), Sungai Buluh Campus, Selangor, Malaysia. 4 Department of

Biochemistry, Medical Faculty, Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia

ABSTRACT

B-cell lymphoma 2 (Bcl-2) family proteins are divided into pro- and anti-apoptotic groups. The

interactions between pro- and anti-apoptotic Bcl-2 family members govern cell survival and death. Cancer

cells overexpressed anti-apoptotic Bcl-2 family members to overcome cell death. The prosurvival Bcl-2

family proteins provide a potential target in cancer therapy. BH3-only proteins are antagonists of

prosurvival Bcl-2 family members. BH3-only proteins trigger apoptosis by binding to prosurvival Bcl-2

family members and releasing proapoptotic Bcl-2 family members from sequestration of the prosurvival

members. Therefore, small molecules (BH3 mimetics) mimicking BH3-only proteins served as promising

anticancer agents. In this study, we examined the potential action of gamma-tocotrienol (a vitamin E

isomer) as an antagonist of Bcl-2 protein in human neuroblastoma SH-SY5Y cells. Our results showed

that gamma-tocotrienol reduced cell viability of neuroblastoma in a concentration dependent manner.

Gamma-tocotrienol induced apoptosis by depolarizing mitochondrial membrane potential and releasing

cytochrome c from mitochondria to cytosol. Caspase-9 and caspase-3 activities were increased by

gamma-tocotrienol while caspase-8 activity was not affected. These results showed gamma-tocotrienol

induced apoptosis by intrinsic pathway. In silico docking analysis suggested gamma-tocotrienol bound to

BH3 domain of Bcl-2 protein. In vitro binding assay demonstrated gamma-tocotrienol was bound to Bcl-2

protein. In conclusion, our data suggested gamma-tocotrienol induced apoptosis by inhibiting Bcl-2

protein function and presented the first report on gamma-tocotrienol with BH3 mimetic-like properties.

YIA-CA2





Silencing of Tousled-like Kinase 1 (TLK1) Reduces Survival, Migration and

Invasion of Glioblastoma multiforme Cells

1,2

Kamariah Ibrahim, 1Mohd Firdaus Che Mat,

1Nor Azian Abdul Murad,

1,3Norfilza Mohd Mokhtar,

1,3Wan Zurinah Wan Ngah,

1,3A Rahman A Jamal and

1,3Roslan Harun

1UKM Medical Molecular Biology Institute (UMBI), Kuala Lumpur, 2Faculty of Medicine, University of Malaya, Kuala Lumpur,

3Faculty of Medicine, National University of Malaysia, Kuala Lumpur.

ABSTRACT

Glioblastoma multiforme (GBM) is an aggressive brain tumour that commonly exhibits resistance to the

conventional chemotherapy and radiotherapy. We had previously performed a high throughput RNAi-

screening and identified Tousled-like kinase 1 (TLK1) as one of the potential therapeutic targets for

GBM. In this study, we investigated the effects of TLK1 silencing on the GBM cells and determined the

molecular mechanisms underlying the cellular dysfunctions. Silencing of TLK1 was performed using

SMARTpool siRNA in both U87MG (PTEN mutant) and LN18 (P53 mutant) cells. The TLK1 loss-of-

function effects were determined using cell viability, apoptosis, clonogenic, invasion and migration

assays. The downstream pathways of TLK1were interrogated using Illumina HumanHT12 v4 BeadChip

microarray. The TLK1 was successfully knocked down at mRNA and protein levels. TLK1 silencing

reduced the cell viability and clonogenic potential of both GBM cell lines. Inhibition of TLK1 also

induced S-phase cell cycle arrest and increased apoptosis. Interestingly, silencing of TLK1 significantly

reduced invasion and migrationof GBM cells. Morphological changes observed in U87MG cells

following TLK1 silencing suggests involvement of actin-cytoskeleton. Silencing of TLK1 in U87MG

cells identified several downstream pathways including cell cycle, focal adhesion and actin cytoskeleton

pathways. Pathway analysis suggested that TLK1 might control GBM cell migration and invasion

through its interaction with the human DEAD box protein p68 in PIK3 pathway dependent manner. In

conclusion, inhibition of TLK1 reduces survival, migration and invasion of GBM cells probably through

the PIK3 pathway.

YIA-CA3





Matrix Attachment Region/Scaffold Attachment Region (MAR/SAR): A Key

Player in Mediating the Bile Acid-Induced Chromosomal Breaks in

Nasopharyngeal Carcinoma (NPC)

SN Tan and SP Sim

Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, Sarawak, Malaysia

ABSTRACT

Various studies have reported that gastroesophageal reflux (GER) may reach nasopharynx. Bile acid

(BA), the main component of refluxate, is a potential carcinogen. BA-induced apoptosis has been strongly

implicated in the aetiology of various malignancies. However, the role of BA in nasopharyngeal

carcinoma (NPC) has not been investigated. Chromosomal breaks occur during DNA fragmentation in

apoptosis and also during chromosomal rearrangements. Chromosomal breaks usually cluster in certain

regions containing specific chromatin structures, such as Matrix Attachment Region/Scaffold Attachment

Region (MAR/SAR). MAR/SAR has been simultaneously implicated in apoptosis and chromosomal

rearrangements. We hypothesised that BA-induced apoptosis may cause chromosomal breaks at

MAR/SAR leading to NPC chromosomal rearrangements. We aimed to compare the cleavage frequency

between SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR) of the

AF9 gene (9p22) upon BA treatment. By using DNASTAR software, MAR/SAR sites in the AF9 gene

were predicted by MAR/SAR recognition signature (MRS). BA treatment was done on NP69 cells. Flow

cytometric apoptosis detection was performed. Nested Inverse Polymerase Chain Reaction (IPCR) was

employed to detect gene cleavage in both the SAR and non-SAR regions. The cleavage frequency of the

SAR region detected in treated cells was significantly higher than that of control (P < 0.05). As for the

non-SAR region, the overall cleavage frequency was two folds higher than that of the SAR region,

however, there was no significant difference between the treated cells and control, suggested that the

cleavages identified in this region were not BA-mediated. By using CENSOR program, we found that

57% of the non-SAR region and 11% of the SAR region have been occupied by repeat elements. It is

likely that the presence of repeat elements leads to the high cleavage frequency in the non-SAR region.

MAR/SAR could be a crucial player in BA-mediated NPC chromosomal rearrangements.

YIA-CA4





Exome Sequencing Identifies Transcription Factors as Commonly Mutated Genes

in Nasopharyngeal Carcinoma

1YP Chow,

2SW Choo,

3PV Lim,

4R Pathmanathan,

1SH Teo

1Cancer Research Initiatives Foundation, 2nd Floor Outpatient Centre, Sime Darby Medical Centre, Selangor, Malaysia, 2Dental Research and

Training Unit, Faculty of Dentistry, University of Malaya,50603 Kuala Lumpur, Malaysia, 3Tung Shin Hospital, Kuala Lumpur, Malaysia, 4 Sime

Darby Medical Centre, Selangor, Malaysia

ABSTRACT

In recent years, utilization of genetic information to stratify cancer patients for treatment with targeted

therapy has achieved remarkable success and improved patients’ survival. In this study, we sought to

identify somatic driver mutations implicated in nasopharyngeal cancer (NPC) tumorigenesis and to

identify potential therapeutic targets, with the hope to expand and guide NPC treatment options. Briefly,

DNA isolated from a total of 10 macrodissected samples (>80% tumor cellularity) and matched blood

samples have been subjected for whole exome sequencing. Sequencing reads were mapped to human

genome hg19 and Genome Analysis Toolkit (GATK) was used to detect single nucleotide variants and

indels. Somatic mutations were identified by excluded polymorphisms found in dbsnp137, 1000 Genomes

Project and 6500 NHLBI exome data, followed by subtraction with variants found in normal blood

samples. A mean coverage of 110X per sample was achieved, with over 98% of targeted bases were

represented by at least 10 reads. We detected a total of 301 somatic mutations, of which 220 were

nonsynonymous (12 indel, 15 nonsense, 6 splicing, 187 missense), and 81 were synonymous. The

analysis revealed genes of several key cancer pathways, including DNA repair, cell cycle regulation,

apoptosis, and immune responses were mutated in NPC. The observed altered pathways are similar to

those identified through gene expression studies, highlighting their essential roles in NPC malignancy

transformation. Also, few cancer census genes were found mutated in NPC. Interestingly, our data

suggests deregulation of regulatory networks may be key driver of NPC tumorigenesis whereby many

transcription factors were found mutated in NPC. In summary, our findings revealed putative driver genes

implicated in both previously unreported and known pathways underlying NPC pathogenesis and

identified new potential candidate driver genes for further characterization.

YIA-CA5





Somatic Mitochondrial DNA Mutations Across Different Grades of Gliomas:

A Whole Genome Resequencing Approach

1 2

BH Soon, 1NA Abdul Murad,

2A Abu Bakar,

2CJ Toh,

2F Fadzil,

2J Thanabalan,

3MS Mohd Haspani,

2R Kumar,

1A Mohd Tamil,

1N Mohd Mokhtar,

1R Harun,

1WZ Wan Ngah,

1R Jamal

1UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Malaysia,

2 Division of Neurosurgery, Department of Surgery,

Faculty of Medicine, Universiti Kebangsaan Malaysia, Malaysia, 3 Neurosurgery Department, Hospital Kuala Lumpur, Malaysia.

ABSTRACT Mitochondria plays an important role in cellular energy metabolism and apoptosis, which are key

components in cancer biology. Mitochondrial DNA (mtDNA) mutations could lead to respiratory chain

dysfunction, affecting tumour growth sustainability and aggressiveness. Our study aim was to compare

somatic mtDNA mutations across various grades of gliomas and to identify and predict their pathogenic

sequelae. In this cross sectional study, 40 glioma tissues of different grades with matched blood samples

were obtained from patients diagnosed with primary brain tumour who underwent surgical excision at the

UKM Medical Centre and Hospital Kuala Lumpur from the year 2010 onwards. Histopathological

examination reports were traced to exclude non-gliomas and for WHO grading classification. Tumour and

matched blood leucocyte DNA were extracted. The entire mitochondrial genome was sequenced using

Affymetrix GeneChip Human Mitochondrial Resequencing Array 2.0 and analysed using GSeq software

to detect sequence alterations. Somatic mutations were identified and compared across grades. All amino

acid changes were analysed for possible pathogenicity of the protein function outcome using PMut and

PolyPhen-2 softwares. A total of 91 point mutations, including 37 novel mutations, were identified in all

samples which were classified into grade 1, 2, 3 and 4 tumours (n=9, 7, 9 and 15 respectively). Overall,

75% of samples harboured at least 1 somatic mutation. High-grade tumors, on average, harbored more

somatic mutations and contained more homoplasmy changes. Conversely, low-grade gliomas had more

missense mutations causing amino acid changes in eloquent coding regions, in which majority of the

affected respiratory chain proteins were predicted as probably damaging or pathological. Proteins of the

respiratory complex 1 and 4 were more frequently affected than others. In conclusion, different grades of

gliomas demonstrated different patterns of somatic mtDNA mutations, which could affect their

respiratory chain function. The putative role of mtDNA mutation in gliomagenesis should be further

explored.

YIA-CA6





A Gene-Expression Signature as A Predictor of Survival in Colorectal Cancer

1Nurul Ainin Abdul Aziz,

1,2Norfilza Mohd Mokhtar,

1 Roslan Harun,

1Md. Manir Hossain Mollah,

3Isa Mohammed

Rose, 4Ismail Sagap,

5 Azmi Mohd Tamil,

1Wan Zurinah Wan Ngah,

1Rahman Jamal

1UKM Medical Molecular Biology Institute, Kuala Lumpur, Malaysia, 2Department of Physiology, Faculty of Medicine, Universiti Kebangsaan

Malaysia, 3Department of Pathology, UKM Medical Center, Kuala Lumpur, Malaysia, 4Department of Surgery, UKM Medical Center, Kuala

Lumpur, Malaysia, 5Department of Community Health, Faculty of Medicine, Universiti Kebangsaan Malaysia.

ABSTRACT

Histopathology assessment is inadequate to predict disease progression and clinical outcomes in

colorectal cancers (CRC). Patients may experience different clinical outcomes although they are in the

same stage of cancers. A more specific and sensitive biomarkers to determine the patient’s survival are

needed. The aim of this study was to determine gene expression signatures that could predict the survival

of patients with colorectal cancers. We examined microarray gene expression profiles of 78 FFPE

colorectal tissues samples using the whole genome cDNA-mediated Annealing, Selection, extension and

Ligation (DASL) assay (Illumina, USA). The gene expression data was analysed using GeneSpring GX

12.0.2. Outliers were detected and automatically replaced with robust means. The normalised expression

values were randomly divided into training and test sets. Hundred different data sets of training and test

sets were generated randomly and three different statistical tests (SAM, LIMMA and t-test) were applied

to identify the differentially expressed genes. There were 16 significant common genes identified from

the permutation, namely Gene 1 to 16. This 16-gene signature was able to significantly predict the

survival of patients with CRCs compared to the conventional Dukes’ classification in both training

(p=1.603e-09) and test set (p=3.675e-10). The performance of this signaturewas further validated as a

significant independent predictor of survival (p=0.01) in independent external validation datasets from

Australian cohort (n=185). In conclusion, profiling of these 16 genes may provide a more accurate

method to predict survival of patients with CRCs and assists in identifying patients who require more

intensive treatment.

P1





Gene Expression Profiling: A Novel Blood Based, 5-Gene Biomarker Panel to

Differentiate Between High and Low Grade Gliomas

SN Ponnampalam, MF Zulkifle, NA Azami, S Ponnusamy, NR Kamaluddin and Z Zakaria.

Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia

ABSTRACT

Brain tumours are the 10th most common cancer in Malaysia (National Cancer Registry, 2006). Gliomas

are the most common of the primary brain tumours and are highly malignant. Several genetic mutations

are associated with gliomas but to date none have shown any significant diagnostic or prognostic value.

In our present study, we have undertaken gene expression profiling in order to develop a panel of genes

that could be used as a potential blood biomarker to differentiate between high and low grade gliomas.

Blood samples were obtained from adult brain tumour patients diagnosed with a glioma. Ten samples

each were obtained from patients with high and low grade tumours respectively. The tumours included

Grades II and III oligodendroglioma and Grades I to IV astrocytoma including glioblastoma multiforme.

Total RNA was isolated from each sample after which first and second strand synthesis was performed.

The resulting cRNA was then hybridized with the Agilent Whole Human Genome (4x44K) microarray

chip according to the manufacturer’s instructions. Samples and Universal Human Reference RNA were

labeled with Cy5 CTP and Cy3 CTP respectively. Microarray data were analyzed by Agilent Gene Spring

12.1V software using stringent criteria which included at least a 2-fold difference in gene expression

between high and low grade samples and statistical analysis using the unpaired student T-test with a p-

value < 0.01. The gene expression profiling indicated that 2,745 genes were differentially expressed with

more than a 2-fold change (p<0.01) between the high and low grade tumours. Of these, 2107 genes were

upregulated and 638 genes were downregulated. We finally selected a panel of 5 genes based on the

stringent criteria above as a potential blood based biomarker to differentiate between the 2 tumour

subsets. This study has identified a novel blood based, 5-gene biomarker panel that can potentially be

used to differentiate between high and low grade gliomas which has future diagnostic and prognostic

value.

P2





Unveiling the role of specific microRNAs and their potential use as anti-cancer

biotherapeutics

*L.L.A. In, N. Othman, L.H. Yap, S.H. Yap, N.H. Phuah, C.S. Ho and N. Hasima.

Institute of Biological Sciences (Genetics & Molecular Biology), Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia.

ABSTRACT

MiRNAs are small non-coding RNAs of about 19-23 nucleotides long that regulate gene expression post-

transcriptionally by either inhibiting mRNA translation or by inducing mRNA degradation. Recently,

state of the art technologies are being developed at an astounding rate which includes miRNA expression

vector systems to boost miRNA levels, as well as anti-sense RNA or RNA interference platforms

designed to knockdown miRNA expression. In this study, the role of specific miRNAs involved in cancer

progression and treatment were unveiled and exploited for their potential use as bio-therapeutics.

Identification of responsive miRNAs in human prostate, cervical and NSCLC cell lines towards cis-

platinum drug treatments, apoptosis-induction stimuli, tumor suppressor gene expressions and variations

between high and low metastatic cancer sub-cell lines were determined using the Affymetrix® GeneChip

®

miRNA array platform, while resulting fold changes were validated using RT-qPCR. In silico target

predictions and gene enrichment analyses were conducted using TargetScan Human v6.2 and DAVID

v6.7 respectively. Manipulation of miRNA levels were performed by transfecting cells with miRNA

mimics to increase specific miRNA levels and with anti-sense oligos to silence specific miRNA levels.

Responses following transfection were evaluated using MTT assays, flow cytometry, Matrigel™ invasion

assays, scratch assays, HUVEC angiogenesis assays and Western blotting. MiRNAs involved in Bcl-xL

silencing were hsa-miR-181a, hsa-miR-769-5p, hsa-miR-361-5p, hsa-miR-1304, and hsa-miR-608, while

those involved in metastatic properties were hsa-miR-92B, hsa-miR-378 and hsa-miR-1827. Meanwhile,

miRNAs found to be involved in cis-platinum drug susceptibility were hsa-miR-138, hsa-miR-210, hsa-

miR-224 and hsa-miR-744, while those affected by the over-expression of the ANXA7 tumor suppressor

gene were hsa-miR-874, hsa-miR-1284, hsa-miR-543 and hsa-miR-409-5p. Therefore, we suggest that

these specific miRNAs do play an important role in modulating responses toward anti-cancer drug

resistance, apoptosis and metastasis occurrences, and that the controlled manipulation of these epigenetic

elements could contribute towards the management of various malignancies.

P3





Hemoglobin Lepore Cases Reported in the IMR

*NA Aziz, P Yelumalai, FA Ab Hamid & R Ahmad.

Molecular Genetics Laboratory, Hematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), National Institute of Health

(NIH), Ministry of Health

ABSTRACT

Hemoglobin Lepore (Hb Lepore) is a type of rare hemoglobin variant which results from unequal

crossing over between the delta and beta globin genes. In addition, Hb Lepore is important because of the

possibility of interaction with beta-thalassemias. Currently, there are limited reports about this rare

disorder in Malaysia. Here we present findings from our routine DNA analysis of beta-globin gene in

Molecular Genetics Laboratory of IMR. Peripheral bloods from seven (7) index cases were sent to our

laboratory for DNA analysis of beta-globin gene. All the referring hospitals were from Peninsular

Malaysia (West Malaysia). Genomic DNA was isolated from the peripheral blood and multiplex ligation-

dependent probe amplification (MLPA) was performed to detect the presence of deletion in the beta-

globin gene cluster. The types and breakpoints of deletion were verified using multiplex-gap PCR and

sequencing. All the index cases referred to us were aged between 5-58 years old. Four patients diagnosed

as carriers or traits were noted to have Hb level ranging between 10-14g/dL, MCV 67-79fL, MCH 28-

33g/dL and slightly high HbF. One homozygote Hb-Hollandia presented as thalassemia intermedia

showed Hb level of 10.5g/dL, MCV 61.6fL, MCH 22.70, and high HbF;79.5%. Two compound

heterozygote of Hb Lepore-beta thalassemia showed low level of hemoglobin; <8g/dL, MCV <63fL,

MCH <21g/dL and HbF <40%. Hemoglobin Lepore trait patients in this study had blood counts which

could not be distinguished from those of beta thalassemia trait. The hemoglobin Lepore homozygote and

the compound heterozygotes for hemoglobin Lepore and beta thalassemia patients presented with the

clinical and haematological picture of thalassemia intermedia. Other studies have reported compound

heterozygotes may have the clinical and hematological picture of thalassemia major or intermedia. Thus

patients who are carriers of this trait should be counselled and advised on risks of conceiving a child with

a transfusion dependent condition.

P4





Role of P-Glycoprotein in Preventing Helicobacter pylori Carcinogenicity

M.Omar

Faculty of Pharmacy, National University of Malaysia, Kuala Lumpur Malaysia

ABSTRACT

Helicobacter pylori infection is the primary cause of gastric cancer. It is believed that P-glycoprotein

expression may be manipulated to prevent H. pylori attachment to the human intestinal epithelium and

limit its potential to cause gastric cancer. Our study aimed to investigate the extent of H. pylori

attachment to the human gastrointestinal cell lines in the presence of a P-glycoprotein potent inducer or

inhibitor. Caco-2 and LS174T cell lines were used to assess bacterial attachment of H. pylori strains (G27

and J99) to see how well they attached to the gastrointestinal cell lines within certain incubation periods.

Valspodar (PSC-833) was used to evaluate whether inhibition of P-glycoprotein would have any

influence on bacterial attachment. The level of P-glycoprotein expression in the LS174T cell lines was

induced with rifampicin for the bacterial attachment study. All bacteria used in the study showed an

increasing pattern of attachment to the Caco-2 cells over a 4 hour study period. H. pylori G27 and J99

demonstrated significantly higher bacterial attachment to Caco-2 cells when PSC-833 was introduced. In

contrast, the bacterial attachment of both H. pylori G27 and J99 to LS174T cell lines were unaffected by

P-glycoprotein expression. Manipulating P-glycoprotein expression by the use of inducers and inhibitors

may influence the H. pylori attachment to gastrointestinal cell lines, consequently altering the risk of

gastric cancer.

P5 Asia-Pacific Journal of Molecular Medicine 2013, 3 (SUPP 1)




Epigallocatechin-3-Gallate Synergizes with Hydroxy-Chavicol to Enhance

Cell Apoptosis in Glioma In Vitro

1Amirah Abdul Rahman*,

1A. Rahman A. Jamal,

2Suzana Makpol,

1Roslan Harun,

1Norfilza Mohd Mokhtar,

1,2Wan

Zurinah Wan Ngah

1UKM Medical Molecular Biology Institute (UMBI), UKM Medical Centre (UKMMC), Cheras, 56000, Kuala Lumpur; 2Department of

Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Aziz 50300, Kuala Lumpur.

ABSTRACT

The phytochemical of green tea, epigallocatechin-3-gallate (EGCG) and hydroxy-chavicol (HC), another

natural bioactive from Piper betle leaves both display potent anticancer activity affecting multiple

signalling pathways. But whether combining them would be an effective therapeutic strategy for brain

cancer was yet to be determined. EGCG, HC and their combination were tested for cytotoxicity on glioma

cell lines 1321N1 (grade II), SW1783 (grade III) and LN18 (grade IV) by cell proliferation (MTS) assay,

and the interaction of EGCG and HC were evaluated through the use of an isobologram. Furthermore, the

effects of these bioactives on the mode of cell death were determined by the presence of active caspase-3

and the staining of annexin V/FITC-PI. Individually, both EGCG and HC reduced the proliferation of

glioma cell lines with high estimated IC50 values ranging from 82-300 µg/ml and 75-119 µg/ml

respectively. Indeed, the combination of EGCG and HC significantly enhanced the inhibition of glioma

cancer cell proliferation than either bioactive alone, and displayed strong synergistic effect on SW1783

with CI of 0.55, and CI=0.53 for LN18. While in 1321N1 cells, moderate synergism interaction of

EGCG+HC was observed with CI value of 0.88. Exposure of grade II, III and IV glioma cells to

combined treatments for 24 hours promotes cell apoptosis as determined by annexin V-FITC/PI staining

and active caspase-3 apoptosis assay, showing caspase-3 activation of 52%, 57% and 9.4% respectively.

Our findings suggest that combined low dose treatment of EGCG with HC may have a potential value in

the treatment of glioma cancer.

.

P6





Aberrant Expression of Collagen Triple Helix Repeat Containing-1 (CTHRC1)

Associated with Pattern of Invasion and Poor Prognosis in Oral Squamous Cell

Carcinomas (OSCC)

1,2*

CE Lee, 1,2

VK Vincent-Chong, 1,2

A Ramanathan, 1,2

TG Kallarakkal, 1LP Karen-Ng,

1WMN Ghani,

1,2ZAA

Rahman, 1,2

SM Ismail, 3MT Abraham,

3KK Tay,

3WMW Mustafa,

4SC Cheong,

1,2*RB Zain

1Oral Cancer Research and Coordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia; 2 Department

of Oral-Maxillofacial Surgical and Medical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia; 3Oral Health Division, Ministry of Health, Putrajaya, Malaysia; 4Oral Cancer Research Team, Cancer Research Initiatives Foundation (CARIF),

Selangor Darul Ehsan, Malaysia

ABSTRACT

Collagen triple helix repeat containing-1 (CTHRC1) is a 30kDa pro-migratory secreted protein. That

inhibits collagen expression and increase cell migration. Elevated expression of CTHRC1 has been

frequently observed in various types of human solid cancers. However the expression and correlation of

CTHRC1 with clinico-pathological parameters in oral cancer remains unclear. Hence, this study aims 1)

to determine mRNA and protein expression levels of CTHRC1 in OSCC compare to normal. 2) To

evaluate the clinical and prognostic significance of this gene in OSCC. mRNA expression level of

CTHRC1 was determined by real time quantitative PCR technique. Protein expression of CTHRC1 in

OSCC and normal tissues was examined using immunohistochemistry (IHC) staining assay. The

association between CTHRC1 and clinico-pathological parameters were evaluated by univariate and

multivariate logistic regression analyses. Overall survival was analysed using Kaplan-Meier analyses and

multivariate Cox regression model. The over-expression of CTHRC1 was found in 88.44% of the OSCC

samples with an average 12.27 fold increases at the mRNA level. Immuno-histochemical analysis showed

that over-expression of CTHRC1 was significantly (p = 0.011) associated with pattern of invasion in

OSCC. The association between CTHRC1 expressions with pattern of invasion remained significant in

multivariate analyses (p = 0.010). Kaplan-Meier survival analyses demonstrated that patients with over-

expression CTHRC1 were significantly associated with poor prognosis (p = 0.002). Adjusted multivariate

Cox regression model revealed that over-expression of CTHRC1 remained as an independent significant

prognostic factor in OSCC (p = 0.019). Current study has demonstrated CTHRC1 expression was

significantly over-expressed in OSCC compared to Normal. Over-expression of CTHRC1 protein could

be an independent predictor for non-cohesive type pattern of invasion and poor prognostic in OSCC.





Modulation of Protein Expression in Signalling Cascades by Tocotrienol Rich

Fraction (TRF) Using Oxidative Stress-Induced C. elegans

Goon Jo Aan, Mohd Shahril Aszrin Zainudin, *Mardiyanna Mohd Adi

Biochemistry Department, Faculty of Medicine, National University of Malaysia

ABSTRACT

Oxidative stress occurs when there is interference on the antioxidant defense system and the production of

free radicals. The aims of this study are to determine the effects of TRF on the protein expression in

signalling cascade as well as to see the effect of TRF on life expectancy and biomarkers of oxidative

stress-induced C.elegans. In this study, C.elegans was induced with 0.3 mM hydrogen peroxide (H2O2)

and treated with 50 µg/ml tocotrienol rich fraction (TRF). The results showed that H2O2 significantly

shorten the lifespan of C.elegans, whereas treatment with TRF significantly increase lifespan compared to

the control. Pre-treatment as well as the combination of pre- and post-treatment of TRF restore the

lifespan of oxidative stress-induced C.elegans to the control. Neither TRF treatment nor H2O2 induction

gave significant effect on protein carbonyl formation in all groups of treatment. The accumulation of

lipofuscin significantly increase in H2O2-induced group as compared to the control, while the pre-

treatment, post-treatment as well as a combination of pre- and post-treatment of TRF showed a

significantly decrease of lipofuscin accumulation as compared to H2O2-induced group. TRF showed no

significant difference to the total number of progeny produced in each group. However, the differences

can be seen on each days of adulthood. Our analysis of protein using two-dimensional electrophoresis

showed 35 proteins were differentially expressed. A total of six proteins were differentially expressed in

TRF treatment group and five in the H2O2-induced group as compared to the control group. Five proteins

in pre-treatment group, nine proteins in the post-treatment group and 10 in a combination pre- and post-

treatment TRF were differentially expressed as compared to H2O2-induced group. There are two proteins

that were differentially expressed in both post-treatment and a combination of pre- and post-treatment of

TRF against H2O2-induced group. From 35 proteins that show differences in expression, only 12 proteins

were successfully identified using MALDI-TOF mass spectrophotometer. Most of this protein such as

mRNA cap guanine-N7 methyltransferase, inorganic pyrophosphates, enoyle co-A hydratase and

triophosphatase isomerase that are involved in the metabolic pathway. In conclusion, TRF modulates the

proteins expression and oxidative stress biomarkers in oxidative stress-induced C.elegans.





Molecular Regulation of FAU in Hydroquinone-Induced Apoptosis

*Siew El,

*Chan Km,

*Rajab Nf,

+Williams Gt,

++Ross D

and

*#Inayat-Hussain Sh

* Faculty of Health Sciences, #Environmental Health and Industrial Safety, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz,

Kuala Lumpur, 50300, Malaysia, +ISTM and School of Life Sciences, Keele University, Keele, ST5 5AZ, UK , ++Department of Pharmaceutical

Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, 80045, USA

ABSTRACT

Hydroquinone (HQ) being a major metabolite of benzene has been shown to induce oxidative DNA

damage leading to apoptosis. This study was conducted to address the potential role of a tumor suppressor

gene Fau (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed

gene) in HQ-induced apoptosis. In this study, we utilized stably transfected cells, rfau (antisense of Fau)

and vector control pcDNA3.1 cells. Apoptosis analysis by externalization of phosphatidylserine

demonstrated that rfau rendered protection on W7.2 towards HQ-induced apoptosis. Moreover, HQ

treatment did not cause an increase of oxidative stress and DNA damage as assessed using

dihydroethidine (HE) staining and Alkaline Comet assay respectively in W7.2 rfau cells. Further

investigation revealed that NAD(P)H: quinone oxidoreductase 1 (NQO1), a detoxification enzyme for

benzene-derived quinones, was upregulated in W7.2 rfau cells. Notably, inhibition of NQO1 by using a

specific mechanism-based inhibitor (MAC220) and NQO1 siRNA re-sensitized W7.2 rfau cells to HQ-

induced apoptosis. Silencing of Fau in W7.2 wild type cells resulted in elevation of NQO1 which further

confirmed the causal link of Fau in the NQO1 expression. Furthermore, our data showed that induction of

NQO1 following downregulation of Fau is via Kelch-like ECH-associated protein-1 (Keap1)/ nuclear

factor erythroid 2-related factor 2 (Nrf2) pathway using immunoprecipitation assay. Moreover, our results

demonstrated that upregulation of glutamate cysteine ligase (GCLC) and accompanied by the increased

GSH level in HQ-treated W7.2 rfau cells. Taken together, this finding provides an insight into the

molecular regulation underlying the role of Fau in the manifestation of benzene toxicity.





Effects of Oxidative Stress on Biochemical Parameters in Breast Carcinogenesis

Ivvala Anand Shaker, Aaminu Ishaka, Kumar Ponnuswamy, Ganesan Kumar, Sakina Roohee, Khaled Saleh.

International Medical School, Management and Science University, University Drive,Off Persiaran Olahraga, Seksyen 13, 40100, Shah Alam,

Selangore Darul Ehsan, Malaysia.

ABSTRACT

Many risk factors have been identified in the causation of carcinoma of the breast, but the mechanisms

by which they increase the risk of the disease are still not clear. Oxidative Stress resulting from an

imbalance between pro-oxidants and antioxidants seems to play an important role in human breast

carcinogenesis. Therefore the present study aimed at confirming the presence of oxidative stress in

patients suffering from carcinoma of the breast and to prove that oxidative stress has a central role and is

the ultimate risk factor among all other risk factors. The study comprised of 50 clinically and

histopathologically proven breast cancer patients in the age range of 25-55 years. The biochemical

estimations carried out in the study were - serum Superoxide dismutase (SOD), Malondialdehyde (MDA),

and Alkaline phosphatase (ALP) levels. The values obtained were compared with age matched equal

number of healthy control female subjects from the same population. Our results showed significantly

higher levels of pro oxidant MDA (p‹0.001) and anti oxidant-SOD enzyme levels (p‹0.001) were found in

the patients group in comparison to control subjects, signifying the presence of oxidative imbalance.

P10





Regulation of TERT Gene in Human T-Lymphoblastoid Cell Line

1*Norodiyah Othman,

2Badrul Hisham Yahaya and

1Zubaidah Zakaria

1Haematology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia, 2Advanced

Medical & Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Pulau Pinang, Malaysia

ABSTRACT

Telomerase enzyme is a ribonucleoprotein complex that is constitutively activated in various types of

cancer cells. The expression of telomerase activity is detected in more than 85% of human tumors

whereas in normal human somatic cells it is either undetectable or present at low levels. There are two

main core component of the human telomerase enzyme; TERT (telomerase reverse transcriptase) and

TERC (telomerase RNA component). Previous investigations have demonstrated that over-expression of

TERT in human malignancies play an important role in maintaining the characteristics of malignant

tumours by having an effect on proliferation, differentiation, and/or immortalization. Thus, inhibition or

over-expression of TERT expression could be a potent therapeutic approach in cancer treatment. In this

study, we introduced TERT siRNA and TERT-DNA clone into the human T-lymphoblastoid cell line

(CCRF-CEM), which had a constitutive effect on activation of telomerase enzymes. The cells were

transfected with TERT siRNA and TERT-DNA clone using transfection reagent (DharmaFect/

Megatran). Real-time PCR was used to study changes in TERT expression levels in transfected cells by

comparing with untransfected (untreated) cells. Knockdown or over-expression of TERT controlled the

expression levels of telomerase enzymes in the cells. Our results demonstrated that TERT played a critical

role in the survival of CCRF-CEM cells, which may have a potential application in designing molecular

therapies for Acute Lymphoblastic Leukaemia treatment.

P11





Significantly Reduced Pro-Apoptotic Protein Bax Expression By Recombinant

Soluble Form Of Heparin-Binding Epidermal Growth Factor-Like Growth Factor

Protein (rsHB-EGFp) Drastically Inhibits Fas-Mediated Fulminant Hepatic Failure

1,2NC Khai,

2,3K Sakamoto,

2K Kosai.

1 International Medical School,, Management and Science University, Shah Alam, Malaysia.2 Department of Gene Therapy and Regenerative

Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. 3 Department of Pediatric Surgery, Kagoshima University Graduate School of Medical and Dental Science, Kagoshima, Japan

ABSTRACT

We have reported that HB-EGF gene is therapeutic for Fas-induced liver failure in mice. But the

molecular mechanism underlying the anti-apoptotic potential of HB-EGF is yet to be investigated. In this

study, we used the recombinant HB-EGF protein and investigated its therapeutic effects and underlying

molecular mechanism. 5- to 6-week-old male C57BL/6J mice (n=8 per group) were administered 3

intraperitoneal injections of 100 μg/mouse rsHB-EGFp at 6 and 0.5hours before and 3 hours after

intraperitoneal injection of 4 μg/ mouse of an agonistic anti-Fas antibody (4Fas-ip). For survival analyses,

8 μg of anti-Fas antibody was injected (n=6). Twenty-four hours after administering of anti-Fas antibody

,the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase

(LDH) levels were remarkably increased in the control mice (2853±814, 1817±469 and 3782±1222 IU/L,

respectively) , but were drastically attenuated to normal levels in the rsHB-EGFp-treated mice (ALT,

21±5; AST, 28±4; LDH, 119±16 IU/L). Accordingly, all of the control mice had histopathological liver

injury, including apoptosis, while none of the rsHB-EGFp treated mice had histopathological findings of

liver injury. Moreover immunoblotting showed that rsHB-EGFp treatment significantly reduced pro-

apoptotic protein Bax expression and no changes in anti-apoptotic protein Bcl-2 and Bcl-XL expression.

In survival analyses, 4 of 6 (67%) control mice died within 17 hours, and only 2 of 6 (33%) mice survived

up to 48 hours. In contrast, all rsHB-EGFp treated mice survived up to 48 hours. Reduced expression of

pro-apoptotic protein Bax by HB-EGF protected liver from Fas-induced apoptosis and fulminant hepatic

failure.

P12 Asia-Pacific Journal of Molecular Medicine 2013




Combination of Nordamnacanthal with Tamoxifen Inhibits Breast Carcinoma

Experimental Tumors by Cell Cycle Arrest and Apoptosis

S. Tamilselvan*, R. Vidhya2, S. Kanagesan

3, S.K. Yeap

4, H. M. Yusof

1, C. P. Osman

5, N. H. Ismail

5, N. B.

Alitheen1

1Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, 43400,

Selangor, Malaysia, 2Department of Oral and Maxillofacial Pathology, Faculty of Dental Science, Sri Ramachandra University, Porur, Chennai,

600116, India, 3Institute of Advanced Technology (ITMA) ,Universiti Putra Malaysia, Serdang, 43400, Selangor, Malaysia, 4Institute of

Bioscience, Universiti Putra Malaysia, 43400 Serdang, Malaysia, 5Faculty of Applied Sciences, Universiti Teknologi Mara, Shah Alam, 40450,

Malaysia.

ABSTRACT

Tamoxifen has proven to exert mainline treatment of breast cancer despite its severe side effects. As an

alternative strategy, we developed a novel combination therapy by combining tamoxifen with

nordamnacanthal. The 4T1 murine breast cancer cells grown both in female BALB/c mice and in culture

were treated with tamoxifen, nordamnacanthal, and the combination of the two drugs in order to

determine treatment efficacies and the mechanism of cell death. The in vivo treatments were evaluated by

monitoring tumor growth and development. The in vitro effects were measured through cell growth

kinetics, cell proliferation and mitochondrial membrane potential disruption assay. The concentration of

tamoxifen needed for an efficient induction of apoptosis in breast cancer cells could be reduced from

21μM to 10μM when combined with nordamnacanthal. A significant (p<0.05) reduction in cell viability

and increase in apoptosis were observed in 4T1 cell lines as compared to vehicle treated controls.

Nordamnacanthal and tamoxifen in combination exhibit a high level of additive effect with enhanced

bioactivity, thereby reducing the required effective dose required for tamoxifen. Our results for the first

time suggest thatcombining tamoxifen with nordamnacanthal can serve as a potential combination therapy

for the treatment of breast cancer with the potential to minimize the side effects associated with high

doses of tamoxifen.





Modeling of Magnetic Nanoparticles' Movement in Capillaries in Presence of

an External Magnetic Field

1Zhila Rashemi,

2*Alireza Bahadorimehr,

3Burhanuddin Yeop Majlis

1Department of Electronics, Islamic Azad University, Arak Branch, Arak, Iran, 2Academic Center for Education, Culture and Research

(ACECR), Arak, Iran, 3Institute of Microengineering and Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia,UKM, 43600 Bangi,

Selangor, Malaysia. Email:[email protected]

ABSTRACT

Microfluidic devices are becoming one of the most dynamic parts of BioMEMS technology. The main

applications of microfluidics are medical diagnostics, genetic sequencing, drug delivery and

hyperthermia. The use of magnetic field for separation of magnetic nanoparticles is a well-known

technique in biology. There are different designs of microfluidic devices that use an external magnetic

field for separation of magnetic nanoparticle in microchannels and capillaries. In this work, we realize the

exact time that magnetic nanoparticles are captured in a capillary under the specific magnetic field to treat

the tumor by drug release or hyperthermia methods. The whole microfluidic device for separation of

functionalized magnetic beads is composed of a fluidic part and a microelectromagnetic coil. The fluidic

chip is a PDMS based device composed of inlet, outlet, microchamber and tubings. The diameter of

nanoparticles that were used in this work are 2.8 μm, 700 nm and 200 nm. Since, the magnetic beads

capturing using electromagnetic coil is concerned, the equations related to electromagnetism, fluids, and

forces on magnetic particles are the most highlighted points of this work. Therefore we model magnetic

nanoparticle motion using COMSOL and calculate the magnetic field that applied on particles. Finally,

we calculate forces on particles, velocity, time constant and trajectory of particles in specific capillary. In

this work, a simple microcoil was used to make the process of capturing and releasing more easily. We

have estimated the time and the place that magnetic particles will be captured in capillaries. This is useful

for tumor treatment in a specified region by drug release or hyperthermia without any damage to other

parts of the body.

P14





Anticancer Activity Of Novel Cell-Penetrating Peptides Against

Breast Carcinoma Cells

1*

Hussin A. Rothan, 2Pottayil G Sasikumar ,

2Ketha Amarnadh Reddy,

3Noorsaadah Abd Rahman and

1Rohana

Yusof

1Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia, 2Department of Medicinal

Chemistry, Aurigene Discovery Technologies Limited, 39-40 KIADB Industrial Area, Electronic City Phase II, Hosur Road, Bangalore 560 100 India, 3 Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT

Cell-penetrating peptides (CPPs) have shown great potential in ligand-mediated drug delivery systems

allowing chemotherapeutic drugs to be more specific to cancerous cells. The general aim of this study

was to design CPPs specific to cancer cells with low toxicity toward normal cells. Short hairpin cationic

peptides (13 amino acids) were designed using protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) as

template. The peptides were synthesized by solid-phase peptide synthesis and disulphide bond formation

followed by peptide purification was confirmed by RP-HPLC and LC-MS. Human breast carcinoma cell

lines (MCF-7) and human normal mammary epithelial cell lines (MCF-10A) were cultured under

standard conditions. Cells viability after treatment with CPPs was measured using MTT assay and cell

population doubling time. Cell membrane integrity was measured by lactate dehydrogenase (LDH)

release from cells with disintegrated membrane and its haemolysis potential was evaluated against human

red blood cells. The ability of CPPs to induce cell death was studied by evaluation of the expression level

of related gene markers using Real time PCR. The data showed that our CPPs (P1 and P2) exhibited low

toxicity against normal cells and low haemolytic potential compared with PG-1. The peptides were more

selective against MCF-7 cells compared to the MCF-10A. The CPPs under study were able to induce

cancer cell senescence and apoptosis in a P53-dependent pathway. These data showed that low molecular

weight peptides as well as alteration of positive charge residues in CPPs reduced their toxicity to normal

cells compared to cancer cells, which is potentially useful to be developed as delivery vectors of

chemotherapeutic anticancer drugs.

P15





Gelam Honey and Ginger Potentiate the Anti Cancer Effect of 5-FU (Fluorouracil)

against Colorectal Cancer Cells, HCT116

1L Hakim ,

1S Makpol ,

1WZ Wan Ngah,

2NA Morad,

1*YA Mohd Yusof

1Department of Biochemistry, Faculty of Medicine; University Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur,

Malaysia, 2Centre of Lipids and Engineering and Applied Research, Universiti Teknologi

Malaysia, Jalan Semarak, 50300 Kuala Lumpur, Malaysia

ABSTRACT

The development of chemopreventive agents using concoction of phytochemicals is potentially viable

approach in preventing and treating colon carcinogenesis. The combined phytochemicals may enhance

multitudes of intracellular effects rather than specific interactions against cancer cells. This study

evaluates the combined anti-proliferative effects of ginger and Gelam honey and its efficacy in enhancing

the anti cancer effect of 5-FU (Fluorouracil) against colorectal cancer cells, HCT116. Cell viability was

measured via MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphenyl)-2H-

tetrazolium) and apoptosis assays. Results showing ginger inhibiting the growth of HCT116 cells much

potently in comparison to Gelam honey with an IC50 of 3mg/mL versus 75mg/mL respectively. Combined

treatment of the two compounds (3mg/mL ginger + 75mg/mL Gelam honey) synergistically lowered the

IC50 of Gelam honey to 22mg/mL. While individual treatment of 5-FU inhibits the growth of HCT116

cells in a dose-dependent manner, the effect was markedly enhanced in combination with 35 mg/mL

Gelam honey. Subsequent analysis on the induction of cellular apoptosis suggests that individual

treatment of ginger and Gelam honey produces higher apoptotic value than 5-FU alone. In addition,

combination treatment of the two natural compounds increases the apoptotic rate of HCT 116 cells dose

dependently while combined treatment with 5-FU only shows modest changes in apoptotic activity.

Combined treatment of Gelam honey and ginger extract enhanced the chemotherapeutic effect of 5-FU

(Fluorouracil) against colorectal cancer.

P16





Zerumbone Loaded Nanostructured Lipid Carrier Regulate the Expression of

Apoptotic Biomarkers in BALB/C Mice Model of Leukemia

1, 2*

Heshu Sulaiman Rahman, 1, 2

Rasedee Abdullah, 2Ahmad Bustamam Abdul,

1Hemn Hassan Othman,

1,2Zeenathul

Nazariah Allaudin, 2Negin Ahmadi

1Faculty of Veterinary Medicine, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia, 2Institute of Bioscience, University Putra

Malaysia, 43400 UPM Serdang, Selangor, Malaysia

ABSTRACT

Zerumbone (ZER) is a natural compound isolated from essential volatile oil of rhizomes of wild ginger

Zingiber zerumbet (L.) smith. The poor water solubility of ZER had limited its therapeutic use. Thus, a

physically stable zerumbone-loaded nanostructured lipid carrier (ZER-NLC) was developed using high-

pressure homogenization (HPH) technique and well characterised by zetasizer, reverse phase high

performance liquid chromatography (RP-HPLC), transmission electron microscopy (TEM), wide angle x-

ray diffraction (WAXR), differential scanning colorimeter (DSC) and Franz Diffusion Cell (FDC) system.

The aim of this investigation is to determine the Bcl-2, Bax, Cyt-c, and PARP gene expression level in

leukemic mice with WEHI-3B cell-induced myelomonocytic leukemia treated orally with ZER-NLC (60

mg/kg) for 4 weeks. Total RNA was extracted from spleen specimens using RNeasy® lipid tissue mini kit

(Qiagen, Valencia, CA). The extracted RNA was quantified using Nanophotometer (IMPLEN, GmbH,

Germany) and was aliquoted and stored at -80˚C. The primer sequences for target (Bcl-2, Bax, Cyt-c, and

PARP) and reference genes (β-actin and GAPDH) were designed and obtained from Integrated DNA

Technologies (IDT, San Diego, USA). QIAGEN® One Step RT-PCR SYBR Green kit (Qiagen,

Valencia, CA) was used to run qRT-PCR according to instructions of the manufacturer. Gradient PCR,

standard curve, and quantitative gene transcription analyses were done using the CFX ManagerTM

software, version 1.6 (BioRad Laboratories, Inc., Hercules, CA) which was provided with the real-time

PCR thermal cycler (BioRad®, CFX96, BioRad Laboratories, Inc., Hercules, CA). As a result, the spleen

cells of ZER-NLC treated leukemic mice showed increased expression of Bax, Cyt-c, and PARP genes

with decreased the expression of Bcl-2 gene. ZER-NLC has excellent potential activity to be developed

for the treatment of cancers especially leukemia.





Differential Effect of Human Mesenchymal Stem Cells on Various Tumours:

Impact on Cell Proliferation and Apoptosis.

*SK Fakiruddin, PJN Baharuddin, MN Lim, NA Fakharuzi and Z Zakaria.

Stem Cell Laboratory, Hematology Unit, Cancer Research Centre (CaRC), Institute for Medical Research (IMR), Kuala Lumpur, Malaysia.

ABSTRACT

Pre-clinical studies demonstrated the anti-tumourigenic effect of mesenchymal stem cells (MSCs) in

several tumor models, while others presented with contradicting evidence. Due to the undefined

connection between MSCs and tumour progression, this study aims to elucidate the anti-tumourigenic and

promoting effect of MSCs on various tumour cells. Isolated MSCs from umbilical cord, bone marrow and

adipose tissue were analyzed for germ layers mRNAs, surface marker expression and mesodermal

differentiation by RT-PCR, fluorescence activated cell sorting (FACS) and histological staining

respectively. Tumor cell lines (HepG2/hepatocellular carcinoma, A549/lung adenocarcinoma, LN-

18/glioblastoma, H2170/lung squamous cell carcinoma, MCF-7/breast adenocarcinoma, REH/acute

lymphoblastic leukemia, K562/chronic myeloid leukemia and KMS-28BM/multiple myeloma) were

cultured in complete RPMI-1640 media. Tumour viability and apoptosis induced by MSCs were

evaluated using methyl-tetrazolium salt (MTS) assay and FACS. MSCs were positive for pluripotent

(Oct-4, Nanog, REX1, and TDGF), germ layer expression (BMP-4, GATA-2, Nestin and SOX-2) and

MSCs surface markers (CD44, CD73, CD105, and CD90). Proliferation of LN-18, H2170 and MCF-7

was significantly inhibited (p<0.001) in cells cultured in 80% MSCs conditioned media. These results

suggest the paracrine effects of MSCs during tumourigenesis. MSCs to tumour ratio (1:5) significantly

(p<0.001) enhanced the proliferation of haematological malignancies, as seen in REH and KMS-28BM

cells. Whereas, significant inhibition (p<0.001) was observed in LN-18 and H2170 tumour cells. Increase

of apoptosis was observed in LN-18, H2170 and MCF-7 cells cultured in 60% MSCs conditioned media

as analyzed using FACS (PI and Annexin V). Our results indicate that MSCs is able to inhibit or promote

tumour growth as shown by the proliferation and apoptosis effects on tumour cell lines. Further in vivo

study is required to verify the role of MSCs in tumour development.

P18





The Role of Multiple Genetic Polymorphisms and Environmental Risk Factors on

Colorectal Cancer Risk in Malaysia

1,2*

N.H. Ramzi, 1J.K. Chahil,

1S.H. Lye,

1K. Munretnam,

1K.I. Sahadevappa,

1S. Velapasamy,

1N.A. Nor Hashim,

3S.K. Cheah,

3G.C.C. Lim,

4H. Hussein,

5M.R. Haron,

1L.W. Ler

and

1L. Alex

1Molecular Research and Service Laboratory, INFOVALLEY® Life Sciences Sdn. Bhd., Unit 1.1, Level 1, Block B, Mines Waterfront Business Park, 43300 Seri Kembangan, Selangor, Malaysia, 2International University of Malaya-Wales, University Malaya City Campus, Jalan Tun

Ismail 50480 Kuala Lumpur, Malaysia, 3Department of Radiotherapy and Oncology, Hospital Kuala Lumpur, Jalan Pahang, 50586 Kuala

Lumpur, Malaysia, 4Department of General Medicine, Hospital Putrajaya, Presint 7, 62250 Putrajaya, Malaysia, 5Department of Radiotherapy and Oncology, Hospital Sultan Ismail, Jalan Persiaran Mutiara Emas Utama, 81100 Johor Bahru, Malaysia.

ABSTRACT

Colorectal cancer (CRC) is second only to breast cancer as the leading cause of cancer-related deaths in

Malaysia. In the Asia–Pacific area, it is the highest emerging gastrointestinal cancer. The aim of this

study was to identify SNPs and environmental factors associated with CRC risk in Malaysia from a panel

of cancer associated single nucleotide polymorphisms (SNPs). In this case-control study, 160 Malaysian

subjects were recruited, including both CRC and controls. A total of 768 SNPs were genotyped and

analyzed to distinguish risk and protective alleles. Genotyping was carried out using Illumina’s

BeadArray platform. Odds ratio (OR), 95% confidence interval (CI) and P-value were calculated using

STATA software. A panel of 23 SNPs that is significantly associated with colorectal cancer risk was

identified (p≤0.01). Among the environmental risk factors investigated, high intake of red meat (more

than 50 percent daily proportion) was found to be significantly associated with increased risk of CRC

(OR=6.52, 95% CI :1.93 - 2.04, P=0.003). Two SNPs including rs2069521 and rs10046 in genes of

cytochrome P450 (CYP) superfamily were found significantly associated with CRC risk. For gene-

environment analysis, the A allele of rs2069521 showed a statistically significant association with CRC

risk when stratified by red meat intake. This study has identified a panel of SNPs that are significantly

associated with CRC in Malaysian population. We also demonstrate an example of genetic risk of CRC

modulated by an environmental factor.

P19





Automated Purification of Total RNA, Including MicroRNA from

Chronic Myeloid Leukaemia (CML) Blood Samples

*

1Aliza MY,

1Chin YM,

1Azli I,

1Nor Asiah M,

2Chang KM,

3Rosline H

and

1Zubaidah Z.

1Institute for Medical Research, Kuala Lumpur, 2Hospital Ampang, Selangor and 3Hospital Universiti Sains Malaysia, Kelantan, Malaysia.

ABSTRACT

Chronic myeloid leukemia gives rise to BCR-ABL fusion gene. MicroRNAs have been identified as

potential indicators for CML. Therefore this study will determine the suitability of methods used to

purify total RNA, including microRNA by comparing quantity of total RNA with regards to number of

white blood cells and quality in patients receiving Imatinib treatment. These usually are within the range

in responders but raised and lower quality in non-responders. Patients were adult aged 18 and above,

either responsive or not to Imatinib treatment, attending haematology clinic in Hospital Ampang,

Malaysia from June to July 2013. Informed consent was obtained and clinical records were examined. A

total of 2.5ml peripheral blood were collected using BD® Vacutainer Safety-Lok™ Blood Collection Set

and drawn into PAXgene® Blood RNA tube. Tube was inverted 10 times, let to stand at room

temperature overnight and stored upright at -200C. Lysate was prepared using PAXgene

® Blood miRNA

Kit followed by automated purification using QIAcube. RNA quantity was measured using NanoDrop®

ND-1000 and quality using Agilent 2100 bioanalyzer with RNA 6000 Nano kit. Out of 18 patients, 12

were responsive to Imatinib treatment and 6 were not. White Cell Count (WCC) was 4.84 to 11.64 with

mean 6.97 x 103/l among responders and 4.32 to 77.94 with mean 20.8 x 10

3/l among non-responders.

Concentration of total RNA were 36.5 to 102.5 with mean 53.4 ng/l among responders and 23.8 to 114.6

with mean 73.4 ng/l among non-responders. rRNA peaks were observed in all responsive samples, only

in 4 among not and 2 showed sign of RNA degradation. Purified total RNA, including microRNA in our

samples showed lower concentration consistent with lower WCC and better quality among responders, in

contrast to among non-responders. Therefore we may consider automated purification of total RNA,

including microRNA carried out as suitable.

P20





Continuous Separation and Enrichment of Circulating Tumor Cells in Blood

Using Lab-On-A-Chip Microfilter

*J. Alvankarian and B. Y. Majlis

Institute of Microengineering and Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

ABSTRACT

Detection and separation of circulating tumor cells in blood is a prognostic and diagnostic way for

metastasis cancers. Due to the low number of CTCs compared to normal blood cell, detection of CTCs is

not easy and takes time. Enrichment of CTCs in conventional labs is achieved by either centrifugation

using density gradient or magnetic separation. These laboratory techniques have long procedures and

CTC identification is normally performed by visual observation of stained cells. The Lab-on-a-chip

technique makes the process of enrichment and separation simpler, faster and at low-cost while it needs

small amount of sample. In this work, development of a microfluidic microfilter for size-based

enrichment of CTCs in human blood, and the potential of this device to allow genomic analysis are

presented. The elastomeric device consists of microstructures with pillar arrays and gaps precisely defined

with microfabrication techniques for capturing the CTCs of size larger than the white bloods cells. The

separation performance on device is investigated with stained blood samples and the fluorescent

microparticles of different size using confocal microscopy. This work presents and validates the present

idea for circulation tumor cell enrichment application. The technique and device demonstrates a high

potential for preparation of the enriched circulating tumor cells for research and clinical studies. By

applying the size-based separation technique between CTCs and human blood cells it is possible to

capture the CTCs on filter with recovery efficiency of above ~90% in the order of few minutes, which is

considerable compared to the present approaches.

P21





Induction of Apoptosis by Hydroxychavicol on HT29, COLO320 and SW837

Human Colorectal Cancer Cells

1*

Khairunnisa’ Md Yusof, 1A Rahman A Jamal,

2Suzana Makpol,


1UKM Medical Molecular Biology, UKM Medical Centre, 2Department of Biochemistry, Faculty of Medicine,

The National University of Malaysia

ABSTRACT

Colorectal cancer is the second most leading cause of death in Malaysia probably due to insufficient

intake of fruits and vegetables as revealed by epidemiological studies. Recently, there is increasing

interest in bioactive compounds found in plants which can modulate the development of cancer.

Hydroxychavicol is a major active compound of Piper betle after eugenol and catechin. Previous studies

showed that hydroxychavicol possess strong anti-mutagenic, antioxidant, and anti-cancer properties. The

aim of this study is to assess the apoptotic effect of hydroxychavicol on three different stages of human

colorectal cancer cell lines HT29, COLO320 and SW837. MTS assay was performed to determine the

cytotoxicity effect of hydroxychavicol (0, 20, 40, 60 and 100 ug/ml) on each cell line for 24 hours. The

half minimal inhibitory concentration (IC50) was obtained and further tests such as annexin V FITC and

active caspase-3 were assessed using flow cytometry with respective dose of hydroxychavicol. The IC50

for HT29 was 40±5.7 ug/ml, COLO320 18±1.2 ug/ml and SW837 30±3.7 ug/ml. For apoptosis

assessment, annexin V FITC showed that hydroxychavicol increased apoptosis in HT29 by 23%, p< 0,05

COLO320 (44.8%, p< 0.05) and SW837 (21.9%, p< 0.05).In addition, flow cytometry anaylsis showed

there was activation of active caspase-3 in cells treated with hydroxychavicol, HT29 (19.7%), COLO320

(11.0%) and SW837 (10.4%) after 24 hours. In conclusion, these findings suggest hydroxychavicol

induced cell death via apoptosis pathway and could serve as a potent anti-cancer agent.





Tocotrienol Rich Fraction Modulate Apoptosis in Glioma Induced in the Mouse

*

aKenny Daun,

bSuzana Makpol,

bNur Fathiah Abdul Sani,

aA. Rahman A. Jamal,

a,bWan Zurinah Wan Ngah

aUKM Medical Molecular Biology Institute (UMBI), Level 7, Clinical Block, UKM Medical Centre (PPUKM) Cheras and bDepartment of

Biochemistry, Faculty of Medicine Universiti Kebangsaan Malaysia, Kuala Lumpur

ABSTRACT

Tocotrienol Rich Fraction (TRF) has potent anti-cancer properties which could be effective for

suppression of glioma. Thus, the study aims to determine the effect of TRF on individual

apoptotic cells in formalin fixed tissue sections of mice brain derived from RCAS/TVA induced

glioma. Plasmid DNA carrying oncogenes of glioma (AKT and KRAS) was transformed into

competent E.coli and the successful transformed plasmid DNA were extracted. The plasmid

DNA was cut by restriction enzyme and then was transfected into chicken fibroblast DF-1 cell.

The transfected DF-1 cell was confirmed by ALV p27 ELISA and western blot. Newborn mice

carrying Nestin/T-va strains were injected with infected DF-1 cell and latency period to develop

glioma was within 2-3 weeks after injection. TRF and stripped oil were administered by force

feeding starting at week 3 and week 7. Hematoxylin and Eosin staining were performed to

identify the morphology changes of the tissue section. Then, TUNEL staining was performed

according to the manufacturer protocol (Roche, Germany) and counter-stained with hematoxylin.

Morphology changes of tissue section were observed and analyzed by determining the staining

colour, size, shape and arrangement of the cells comparing to normal tissue section, untreated

and treated groups. TUNEL staining was analyzed by Image J software. In the mice induced with

glioma, glial cells were poorly defined in size and shape. TUNEL staining results showed that

gliomagenesis resulted in a high number of cells stained with hematoxylin in glioma compared to

normal tissue section indicating the cells did not undergo apoptosis. Treatment with TRF to

glioma induced mice resulted in brown staining indicates the present of apoptotic cells in the

tumor. In conclusion, TRF was able to modulate apoptosis although further analysis is needed to

confirm the involvement of apoptotic signaling pathway.





Identifying Differences in Oxidative Stress in Premature Coronary Artery Disease:

A Preliminary Report

aMuhammad Faizan A. Shukor,

aNoor Akmal Shareela Ismail,

b Rosli Mohd Ali,

b Ika Faizura Mohd Nor,

b Azmi

Mohd Ghazi, cRoslan Harun,

a, cWan Zurinah Wan Ngah.

aDepartment of Biochemistry, Faculty of Medicine Universiti Kebangsaan Malaysia, Kuala Lumpur, b Institut Jantung Negara Kuala Lumpur,

cUKM Medical Molecular Biology Institute (UMBI), Level 7, Clinical Block, UKM Hospital Cheras

ABSTRACT

Oxidative stress is one of the leading causes for coronary artery disease (CAD). CAD

predominantly manifests in older individuals but the number of incidence in younger adults is

increasing. Thus the aim for this research is to determine the oxidative stress status in premature

CAD compared to CAD. Subjects were recruited from Universiti Kebangsaan Malaysia Medical

Centre and Institut Jantung Negara and divided into four groups, i.e., Group I: Healthy control <

45 years old; Group II: Subjects with PCAD < 45 years old; Group III: Healthy control > 60

years old; Group IV: Subjects with CAD > 60 years old. Subjects with CAD were recruited

during angiography procedure while healthy controls were selected based on normal

electrocardiogram (ECG) results. Sixty percents of the subjects with CAD have multiple risk

factors including smoking, hypertension, hyperlipidemia and diabetes mellitus. Our preliminary

results on DNA damage measured by comet assay showed PCAD have significantly higher

damage compared to healthy control within same age group. No significant difference was found

when comparing between subjects > 60 years old with subjects < 45 years old. Antioxidant

enzyme superoxide dismutase (sod) activity showed no significant difference between subjects

and healthy controls within same age group. However, PCAD has significantly higher sod

activity compared to CAD. No significant difference was observed for catalase activity. In

conclusion, our preliminary results suggest that oxidative stress status of a subject could be the

key event leading to CAD.





The Association between Breast Cancer Subtype and TNM Staging: Review of

Database in AMDI, USM

*Nizuwan A, *Nurdianah HF, **MY Musa

*Research Officer, Advanced Medical and Dental Institute (AMDI),USM, Bertam, Penang, **Senior Lecturer, Advanced Medical and Dental Institute (AMDI),USM,Bertam, Penang

ABSTRACT

Breast cancer is the most common cancer in women worldwide, comprising 23% of all female cancers.

Prognosis and survival rates for breast cancer vary greatly depending on the cancer type, stage and

treatment received. The objective in this study is to determine the prevalence of breast cancer subtype and

its association with TNM staging. A total of 236 breast cancer cases from 2004 to 2012 reported to

AMDI, USM were reviewed. The data was categorized into several clinicopathologic factors and their

associations with TNM staging were analyzed using Pearson Chi Square statistical method. The

commonest breast cancer subtype was luminal A with 32.6% of all patients. Triple negative subtype

represents 16.9% of all subtypes and it was significantly associated with first presentation at stage 4 (p-

value <0.05). Majority of the patients presented at Stage 2 (57.2%) followed by Stage 3 (27.1%). Stage 1

and Stage 4 were 11.9% and 3.8%, respectively. The Luminal A breast cancer subtype was the

commonest of all subtypes. Triple negative subtype marked the worst prognosis, recording the highest

number presented at advance stage.


Abstracts for the 1stNational Conference for Cancer Research &


8-10thNovember 2013, The Royale Chulan,Kuala Lumpur

Combining Gene Expression and microRNA Profiling of Human Metastatic

Colorectal Cancer Identifies Putative Gene Targets

1Aimi Syamima Abdul Manap,

2Isa Mohamed Rose,

3Ismail Sagap,

1Manir Hossain Mollah,

1Roslan Harun,

1Rahman Jamaland

1,4Norfilza Mohd Mokhtar

1UKM Medical Molecular Biology Institute, Kuala Lumpur, Malaysia, 2Department of Histopathology, UKM Medical Centre, Kuala Lumpur,

Malaysia, Kuala Lumpur, Malaysia, 3Department of Surgery, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 4Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.

ABSTRACT

Most of cancer deaths are mainly due to the development of tumor metastasis rather than the primary

lesion itself. Roles of microRNAs (miRNAs) in post-transcriptional gene regulation of metastatic genes

are presumed to be extensive. The aim of this study is to identify differentially expressed miRNA and

mRNA of targeted genes in primary colorectal cancer with or without liver metastasis as compared to

their neighboring non-cancerous tissues. Total RNA from twenty-four samples were sent to Exiqon

(Denmark) for miRNA profiling using LNA™ microRNA Array. We have carried out gene expression

profiling by Affymetrix Human Gene 1.0 ST Array using similar samples. To obtain high stringency

results, five statistical test methods were used to analyze the microarray results. We performed MAGIA2

web tool for miRNA-mRNA interactions and identified a total of 109 DE-miRNAs in liver metastases

compared to liver normal where 87 satisfy three of statistical tests and 22 satisfy two of three statistical

tests, 51 down-regulated and 58 up-regulated. We finally demonstrated that 9 significant differentially

expressed miRNAs, miR-301a, miR-203a, miR-150, miR-301b, miR-182, miR-183, miR-181c and miR-

139-5p. The majority of these miRNAs were involved in cancer progression and metastasis pathways

namely angiogenesis, apoptosis as well as p53 signaling pathway. Among these miRNAs, mir-181c was

reported to be involved in breast cancer metastasis, mir-150 in ovarian cancer metastasis and mir-203 in

prostate cancer metastasis. For gene expression profiling, integrative miRNA-mRNA analysis identified

42 targeted genes for these miRNAs in which they were differentially expressed in cancer compared with

non-cancerous samples. We also observed that some of others like NDRG2, ANLN, PDE4D and FOXP2

were among tumor suppressor gene, transcriptional repressor, involved in cellular proliferation and

differentiation in targeted tissues. The discovery of these miRNAs and their targeted genes in CRC

metastasis to liver has provided novel mechanism for regulating human gene expression that impacts

diverse biological and pathological processes.

P26





Evaluation of Gene Expression between Bi-dimensional Monolayers (2D) and

3-Dimensional (3D) Tumor Spheroids of Cervical Cancer Cell Lines

1*

KalaivaniMuniandy, 2Nethia Mohana Kumaran,

3Shaharum Shamsuddin,

1Venugopal Balakrishnan

1Institute for Research in Molecular Medicine (INFORMM),Universiti Sains Malaysia, 11800, USM, Pulau Pinang, Malaysia, 2School of

Biological Sciences, Universiti Sains Malaysia, 11800,USM, Pulau Pinang, Malaysia, 3School of Health Science, Universiti Sains

Malaysia,16150, Kubang Kerian,Kota Bharu, Kelantan, Malaysia.

ABSTRACT

Cells, grown as bi-dimensional monolayers (2D models,) are routinely used as initial model system for

evaluating gene expression level in mammalian cells. The 3D cell culture method shows a high degree of

biological and clinical relevance to in vitro models. Recently, 3D culture gene expression profiles have

been shown to be more accurate, which reflects clinical expression profiles compared to 2D cultures. The

3D cultured cells provide a cellular microenvironment that more closely mimics the microenvironment

observed in native tissue. To obtain an insight into gene expression level of cervical carcinoma cell lines,

HeLa and Caski cells were cultured in bi-dimensional monolayers (2D) and 3D tumor spheroids. 3D

spheroids of HeLa and Caski cells were generated using the liquid overlay culture method. 3D tumor

spheroids were generated upon 72 hours culture in agarose coated plates. Total RNA was isolated from

cells cultivated in 2D and 3D environments. Quantitative real-time PCR analysis of the expression of

CTCF, YB-1, c-myc and HPV E7 genes was performed and differences in gene expression level were

calculated and compared with endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

gene. Monolayer and spheroids culture of human cervical carcinoma cell line cells demonstrates distinct

gene expression pattern. This finding illustrates the importance of physical environment of cellular

organization and effects on gene expression pattern.

P27





Goniothalamin Inhibits Proliferation, Migration and Invasion of

Human Glioma Cells Independent of NQO1 Expression

*

1Ruhana Hamzah,

1Bee Hong Soon,

2Kok Meng Chan,

1Hui-Min Neoh,

1Rahman Jamal,

1,2Salmaan Hussain

Inayat-Hussain

1UKM Medical Molecular Biological Institute, UKM Medical Centre, 2Faculty of Health Sciences, National University of Malaysia

ABSTRACT

Goniothalamin (GTN) has been shown to possess many biological activities including potent cytotoxicity

towards various cancer cell lines. However there is no available information on its anti-cancer effects on

human gliomas cell. Therefore, in this study we have characterized the anti-cancer effects of GTN on

various grades of gliomas cell. Assessment of cytotoxicity using 3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay showed that GTN was cytotoxic towards gliomas cells

whereby the effects was more potent toward the LN-18 cells (Grade 4) as compared 1321 N1 (Grade 2)

and SW 1783 cells (Grade 3). Interestingly, the effects of anti-migration induced by GTN was more

potent towards grade 2 gliomas followed by grade 3 and grade 4. We further performed the measurement

of reactive oxygen species (ROS) and GSH using Mitosox and Ellman's reagent respectively. GTN-

treated cells resulted in the concurrent increase of ROS and decrease of GSH which suggest oxidative

stress mediates GTN-induced cytotoxic and anti-migration effects. Assessments of total antioxidant

potential by Ferric Reducing Antioxidant Power (FRAP) assay reveal higher antioxidant capacity were

detected in higher grade gliomas. This include glutathione (GSH) and NAD(P)H Quinone Oxidoreductase

1 (NQO1) whereby NQO1 may be the one that contribute to this high antioxidant capacity in human

gliomas cell. Taken together these data demonstrated that GTN may serve as potential treatments for

brain cancer in future.

http://en.wikipedia.org/wiki/Di-

http://en.wikipedia.org/wiki/Di-

http://en.wikipedia.org/wiki/Thiazole

http://en.wikipedia.org/wiki/Phenyl

P28





Characterization Of Common Transcription Factor Binding Sites In

Colorectal Cancer Related Genes

1Intan Fairuz Ramli,

1,3Roslan Harun,

1Rahman Jamal and


1UKM Medical Molecular Biology Institute (UMBI), 1,3Department of Medical, 1,2Department of Physiology, Faculty of Medicine,

Universiti Kebangsaan Malaysia (UKM).

ABSTRACT

The incidence of colorectal cancer (CRC) in Malaysia has reached an alarming stage where it ranks the

top most cancer case for males and the third for females. Therefore, we hope to unfold colorectal

carcinogenesis by determining transcription factor binding site (TFBS) within the promoter of CRC

genes. Microarray data from 13 paired samples of normal and CRC Dukes’ B, C and D was statistically

analysed using GeneSpring GX12.5. The differentially expressed genes were grouped according to their

expression pattern using K-means clustering. Using MATCHPRO

(TRANSFAC), cross-cluster analysis

was conducted leading to the identification of the most common TFBS. Both data were analysed using

Pathway Studio to determine the interactions between the TFBS and CRC genes as well as the significant

pathways involved.Within the approximately 2kb length promoter of CRC genes chosen,the functionality

of specific TFBS were assessed using dual-luciferase assays. Once the functional TFBS is identified, loss-

of function mutation will be introduced into the sequence using site-directed mutagenesis to investigate

the role of the TFBS. In this study,the functionality of PAX4 within the promoter of MET, MIF, CSE1L

and TOMM34 were analysed. These genes were significantly involved in cell proliferation pathway

(p=3.26E-06). Preliminary dual-luciferase assay results showed all the promoters were functional when

transfected into HCT116 cell line. The normalised fold change in expression was seen the highest in

CSE1L which was 19.56 followed by MIF, TOMM34 and MET at 8.97, 6.67 and 1.27 respectively.

Previous study has suggested, overexpression of PAX4 could act as tumor suppressor in human

melanoma cells. However the detailed molecular mechanism is still unknown. Though the promoters of

MET, MIF, CSE1L and TOMM34 was observed to be functional, futher elucidation of the specific

location of PAX4 in the promoter region is required to understand its function in regulating colorectal

cancer related genes.

P29

Asia-Pacific Journal of Molecular Medicine 2013




Gene expression of GATA4 and SPP1 revealed signaling pathways in

human cumulus cells

1Muhammad Hamzihadi Hamrah,


2 Rahman Jamal,

3Zainul Rashid Mohd Razi,

3Nurshaireen Che Abdullah

and

1Noor Wahidah Mohd Nasri

*

1Department of Physiology, UKM Medical Centre, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia, 2UKM Medical Molecular

Biology Institute (UMBI), 7th Floor, UKM Medical Centre, Jalan Ya’acob Latiff, 56000 Cheras, Malaysia, 3Department of Obstetrics and

Gynaecology, UKM Medical Centre, Jalan Ya’acob Latiff, 56000 Cheras, Malaysia.

ABSTRACT

About 90% of women who were attended to infertility clinics for anovulatory infertility were diagnosed

with Polycystic Ovary Syndrome (PCOS). Several responsible genes from previous studies have been

identified such as GATA4 and SPP1 that play a role in infertility but still there was no defined gene in

relation with PCOS. However, the clear function and specific pathway involved in these genes have not

yet been studied. Therefore, this research aim to study the expression of GATA4 and SPP1 using

previous microarray data and to suggest a new molecular pathway related to infertility in PCOS. High

RNA quality from human cumulus cells from 16 infertile patients undergoing in vitro fertilization in

Universiti Kebangsaan Malaysia Medical Centre in between 2012 till 2013 were chosen prior to

amplification and hybridization to the Affymetrix GeneChip® PrimeView™ Human Gene Expression

Array. Microarray data were analysed using GeneSpring GX 12.5 and Pathway Studio 9.0 software. The

filtered microarray data imported to pathway studio revealed 95 signaling pathways and 379 biological

processes. The significant pathway related with GATA4 and SPP1 was the Ariadne receptor signaling

pathway. Significant biological processes (p<0.05) detected GATA4 in in utero embryonic development

and SPP1 in anti-apoptosis. In conclusion, we proposed a new discovery role and pathways of VEGFA

and SPP1 in the cell signaling pathway contribute to a new knowledge in understanding infertility in

PCOS.

P30





Evaluation of Biallelic Genetic Markers For Chimerism Analysis In

Malaysian Pediatric Patients

*BL Kueh, R Ahmad, E Esa, H Mohd Ibrahim, M Harun and Z Zakaria

Institute for Medical Research, Kuala Lumpur, Malaysia.

ABSTRACT

Post hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse

and therapeutic intervention. The aim of this study is to comparatively evaluate and identify the most

informative marker in the context of chimerism analysis among the population in Malaysia. We

evaluated 11 biallelic genetic markers (S01 to S11) for their informativity in 64 pediatric patients and

their related donors by using real-time quantitative polymerase chain reaction (PCR) assay. These

biallelic genetic systems were selected from 19 specific sequence polymorphisms (markers) in the human

genome located on 9 different chromosomes. Among these 19 markers, 15 markers were able to

differentiate the 54 recipient donor pairs while there were no informative markers for 10 recipients. The

informativity of these markers were found to be variable, ranging from 0 to 16.7%. Half (50%) of our

patient samples were analyzed with markers S01b, S03, S06 and S08b. This indicated that selection of

suitable markers is important to improve diagnostic testing.

P31





Co-Expression of Epithelial-Mesenchymal Transition Markers LAMC2 and

TWIST1 is Associated with Poor Overall Survival in

Oral Squamous Cell Carcinoma

Y.H. Kong*

1,2, S.N. Syed Zanaruddin

2, A. Ramanathan

1, T.G. Kallarakkal

3, V.K. Vincent-Chong

1, W.M. Wan

Mustafa1, M.T. Abraham

4, S.H. Lau

5, R.B. Zain

1,3, Z.A. Abdul Rahman

1,3 and S.C. Cheong

2,3

1Oral Cancer Research & Coordinating Centre (OCRCC), University of Malaya, Kuala Lumpur, Malaysia, 2Oral Cancer Research Team,

Cancer Research Initiatives Foundation (CARIF), 12A, Jalan TP5, Taman Perindustrian UEP, 47600 Subang Jaya, Selangor Darul Ehsan, Malaysia, 3Department of Oro-Maxillofacial Surgery and Medical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, 4Dept. of

Oral & Maxillofacial Surgery, Tengku Ampuan Rahimah Hospital, Klang, Malaysia, 5Stomatology Unit, Institute for Medical Research, Jalan

Pahang, 50588 Kuala Lumpur, Malaysia

ABSTRACT

Epithelial-mesenchymal transition is one of the major hallmarks of malignancy that contribute towards

cellular migration and invasion in tumorigenesis. Currently, the clinical and pathological implication of

EMT markers at invasive tumor front of oral squamous cell carcinoma remains elusive. We aim to

investigate the expression of EMT markers CDH1, LAMC2, SNAI1/2, ZEB1, ZEB2 and TWIST1 at the

invasive tumor front of oral squamous cell carcinoma (OSCC) by immunohistochemistry and correlate

these expressions with clinical and pathological outcomes. In this study, loss of CDH1, diffused LAMC2,

and expression of SNAI1/2 and TWIST1 were significantly observed in tumor tissues compared to non-

malignant oral mucosa tissues (p < 0.05). Diffused LAMC2 at invasive tumor front was associated with

non-cohesive pattern of invasion (p = 0.014), suggesting LAMC2 plays a role in the dissemination of

cancer cells. TWIST1 expression was associated with worse overall survival of patients (p = 0.035),

however, co-expression of diffused LAMC2 with TWIST1 showed stronger association with worse

overall survival of patients as compared with TWIST1 alone (p = 0.011). Taken together, this data

demonstrates that LAMC2 and TWIST1 could be important drivers in OSCC development and both could

work in concert in conferring aggressive disease in OSCC.

P32





Constructing the Cancer Progression Model: Identification of Key Players in

Malignant Transformation

*

1SN Syed Zanaruddin,

1,2IKN Chiang,

1CP Gan,

1Z Fadlullah,

1,2KR Dionne,

3MT Abraham,

4T.G. Kallarakkal,

4ZA

Abdul Rahman, 1,4

SC Cheong

1Oral Cancer Research Team, Cancer Research Initiatives Foundation (CARIF), 12A, Jalan TP5, Taman Perindustrian UEP, 47600 Subang

Jaya, Selangor Darul Ehsan, Malaysia, 2Oral Cancer Research and Coordinating Centre, University Malaya, Kuala Lumpur, Malaysia, 3Dept. of Oral & Maxillofacial Surgery, Tengku Ampuan Rahimah Hospital, Klang, Malaysia, 4Department of Oro-Maxillofacial Surgery and Medical

Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia

ABSTRACT

It is well established that cancer occurs as a result of a sequential accumulation of genetic alterations.

Often, although not obvious, cancer is preceded by a pre-malignant stage. The identification of distinct

genetic profiles of different stages of the tumor paves an opportunity for cancer therapeutic intervention.

Using oral squamous cell carcinoma (OSCC) as a model, we aim to delineate key players in malignant

transformation. In a pilot study, we have successfully established a series oral mucosa cell line

comprising of normal, oral potentially malignant disorder (OPMD) and OSCC from the same patient.

This patient presented with a concurrent OPMD at OSCC diagnosis. We examined the growth

characteristics, molecular changes by RNA sequencing (Illumina HiSeq2000/2500) and invasion ability

by organotypic culture of each of the three cultures. Both the OPMD and OSCC cultures had similar

population doubling (PD) time of 5-6 days. Preliminary RNAseq analysis revealed that mutations in

genes implicated for head and neck cancer such as TP53, NOTCH1, HRAS and PIK3CA were already

present in pre-malignant state suggesting that key molecular aberrations occur before the development of

malignancy. Three-dimensional organotypic culture of these lines demonstrated that the OSCC line

exhibited an invading morphological features as compared to the normal and OPMD line. These initial

data provide insights into the genetic features underlying oral cancer progression and further

characterization of this model could lead to the development of an essential model for the study of

malignant transformation in cancer development.

P33





The Molecular Bases for Beckwith-Wiedemann Syndrome and Russell-Silver

Syndrome.

1*

T Krishnan, 1R Poh,

2T Ishak,

3HB Chew,

3GS Ch’ng,

3WT Keng,

2MK Thong

1 Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia, 2 Department of Paediatrics,

Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia, 3 Department of Genetics, 50586 Hospital Kuala Lumpur, Malaysia.

ABSTRACT

Beckwith-Wiedemann (BWS) and Russell-Silver syndromes (RSS) are clinically and genetically

heterogeneous syndromes which cause gigantism and growth retardation, respectively. BWS is also

known to be associated with increased risk of childhood cancers. Mutations, genomic imprinting errors

and uniparental disomy in chromosomes 7 and 11 have been reported before. The imprinted 11p15 region

consists of two imprinted domains: imprinting centre (IC) 1 which regulates the expression of IGF2 and

H19, and IC2 which controls the expression of CDKN1C , KCNQ1OT1 and KCNQ1. We aim to study the

molecular basis for BWS and RSS. We report the preliminary results of our analyses. Whole blood

samples were collected from 8 BWS and 15 RSS patients based on clinical proforma and the genomic

DNA was extracted. The DNA samples were subjected to multiplex ligation-dependent probe

amplification (MLPA) to detect the copy number status in imprinted genes of chromosome 11 for BWS

and RSS. Two designated controls were included as reference. The resulting PCR products were sent for

fragment analyses. With BWS, all 8 samples carried duplication involving IC2. KCNQ1OT1 is a

paternally expressed untranslated transcript, read in an antisense direction with respect to KCNQ1 which

is required for silencing of imprinted genes. CDKN1C is a maternally expressed imprinted gene encoding

a cyclin‐dependent kinase inhibitor that regulates prenatal and postnatal growth and development. With

RSS, 4 of the 15 samples carried duplications in both IC1 and IC2, whereas 11 showed duplication in IC2

alone. In addition, 1 patient had mutations in all the studied genes in the IC1 and 1C2 regions; 1 patient

showed mutation of 1 gene each at IC1 and IC2; and 3 patients showed mutations at IC2 alone. The

patients in this study present with duplication in BWS, and duplication and mutation in RSS, in different

imprinted genes, emphasizing the heterogeneity of the two growth syndromes. Further confirmation is

ongoing.

P34





Identification of Immunogenic MAGED4B Peptides for Vaccine Development in

Oral Squamous Cell Carcinoma

*

1KP Lim,

1,4AL Chun,

1CP Gan,

1SH Teo,

2ZA Abdul Rahman,

3MT Abraham,

4RB Zain,

5S Ponniah and

1,2SC

Cheong


Jaya, Selangor Darul Ehsan, Malaysi, 2Department of Oro- Maxillofacial Surgery and Medical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, 3Department of Oral & Maxillofacial Surgery, Tengku Ampuan Rahimah Hospital, Klang, Malaysia, 4Oral Cancer

Research and Coordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia, 5Cancer Vaccine

Development Program, Uniformed Services University of the Health Sciences, Bethesda, USA

ABSTRACT

Peptide vaccine is an immunotherapeutic strategy that utilizes tumour specific peptides to illicit an anti-

tumour response. A key step in the development of peptide vaccines is the identification of tumour-

specific proteins. We have previously demonstrated that the melanoma antigen, MAGED4B, is over-

expressed in oral squamous cell carcinoma (OSCC) and this expression promotes proliferation and cell

migration. In this study, we have identified 9 short peptides specific to the MAGED4B protein that are

restricted in binding to the HLA subtypes common in Asians (HLA-A2, A11 and A24). Several in vitro

assays were conducted to evaluate the performance and efficacy of these peptides. The binding, dimer and

ex vivo ELISpot assays demonstrated that the peptides had good binding affinity with the MHC-Class I

molecules and were immunogenic. IFN gamma and Granzyme B ELISpot assays demonstrated that T

cells stimulated with peptide-pulsed dendritic cells had enhanced T-cell cytotoxic activity against

MAGED4B-expressing oral cancer cell lines. Taken together, these MAGED4B-specific peptides could

induce anti-tumour immune responses suggesting that they could be further developed as vaccine

candidates for the treatment of OSCC.

P35





Characterization of Oral Cancer Cell Lines in Xenograft Mouse Models

*1C. P. Gan,

1K. K. Sam,

2V. Patel, T. G. Kallarakkal

3,

3, 4R. B. Zain,

5S. H. Lau,

2A. Molinolo,

2J. S. Gutkind, and

S. 1, 3

C. Cheong


Jaya, Selangor Darul Ehsan, Malaysia. 2Oral and Pharyngeal Cancer Branch, National Institutes of Dental and Craniofacial Research, National

Institutes of Health, Bethesda, USA. 3Department of Oro-Maxillofacial Surgery and Medical Sciences, Faculty of Dentistry, University of

Malaya, Kuala Lumpur, Malaysia. 4Oral Cancer Research and Coordinating Centre (OCRCC), University of Malaya, Kuala Lumpur, Malaysia. 5Stomatology Unit, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia

ABSTRACT

Preclinical use of mouse models is essential for the understanding of tumour biology and drug response.

We have established a large panel of oral cancer cell lines that have molecular characteristics that closely

resembled that of the origin tumour tissues. In this study, we characterized these cell lines in the

subcutaneous and tongue-orthotopic xenograft mouse models. Seven oral cancer cell lines were

maintained in complete DMEM/F12 medium. NU/NU mice at 6 weeks of age were used in the

subcutaneous xenograft model. Oral cancer cell lines (2x106) were injected into the lower flank of the

animal. Meanwhile, SCID/NOD mice at 6 weeks of age were used in the tongue-orthotopic xenograft

model. 1x105 cells were injected into the lateral border of the tongue, under anesthetic condition. All

animals were monitored for general well being and tumour growth. Animals were euthanized and the

tumours were harvested. Cervical lymph nodes in the tongue-orthotopic model were also collected for

histological analysis. ORL48, ORL115 and ORL150 were tumourigenic in the subcutaneous model. Oral

cancer cell lines showed enhanced tumourigenicity when xenografted onto the tongue-orthotopic model

where 6/7 cell lines formed tumours (ORL48, ORL115, ORL150, ORL136, ORL174, ORL204). Of the

cell lines that formed tumours in the orthotopic model, lymph nodes metastasis was observed in ORL48,

ORL115 and ORL150. Notably, the histology of the xenograft tumours closely resemble that of the

corresponding patients’ tumour. The characterization of these cell lines in xenograft models will serve as

important tool to help us identify key drivers of OSCC and more importantly will be useful in the

development of molecular-targeted strategies to combat oral cancer.





The Prevalence of Germline TP53 Mutation in Malaysian Early-Onset

Breast Cancer Patients

1*WX Wen,

1HN Hasmad,

1DSC Lee and

1,2SH Teo

1Cancer Research Initiative Foundation, Sime Darby Medical Centre, Subang Jaya, Selangor, Malaysia, 2Breast Cancer Research Unit,

University Malaya Medical Centre, University Malaya Cancer Research Institute, Kuala Lumpur, Malaysia

ABSTRACT

Breast cancer is the most common cancer among Malaysian women. The crude incidence is estimated to

be 34.86 per 100,000 population. Germline TP53 mutations predispose women to early-onset breast

cancer and are associated with Li-Fraumeni syndrome, an autosomal dominant hereditary cancer disorder

predisposing to a diverse range of neoplasms including breast carcinomas, soft-tissue sarcomas,

osteosarcomas, leukemia, brain tumors and adrenocortical carcinomas. A total of 144 patients with early-

onset breast cancer (≤ 35 years) treated at University Malaya Medical Centre (UMMC) and Sime Darby

Medical Centre (SDMC) between 2003 and 2013, were recruited and tested for TP53 germline mutations

by direct DNA sequencing. We identified seven TP53 germline carriers among the early-onset breast

cancer patients and six exonic variants in TP53. Exonic variants E346X, G334_R335dup6, R273H and

D41fs*3 are clinically relevant while missense mutations A138V and E285K are likely to be clinically

relevant, on the basis of co-segregation and loss of heterozygosity (LOH). Clinically relevant TP53

germline mutations were identified in 5 of the 8 patients (63%) from LFS/LFL families; 1 of the 18

patients (6%) with family history of breast cancer only; and 1 of the 118 patients (<1%) who are not from

LFS/LFL families or with no family history of breast cancer only. Furthermore, these mutations were

found in 5 of the 14 patients (36%) with bilateral breast cancer and 7 of the 59 patients (12%) with human

epidermal growth factor 2 (HER2) positive breast tumors. Our study reports germline TP53 mutations are

detected in 5% of Malaysian early-onset breast cancer patients, suggesting that TP53 mutation screening

should be considered for these patients. Additionally, having a family cancer history that meets LFS/LFL

criteria, bilateral breast cancer and HER2/neu positive tumor are strong predictors for germline TP53

mutation.





The Use Of In Vitro Models for Modeling Drug Sensitivity

*1

P. S. Yee, 1,2

K. N. Ivy Chiang, 1,2,3

K. R. Dionne, 1M. Z. H. Fadlullah,

1K. B. Bernard Lee,

5M. T. Abraham,

6S. M.

Ismail, 6Z. A. A. Rahman,

1,6S. C. Cheong.


Jaya, Selangor Darul Ehsan, Malaysia, 2Oral Cancer Research and Co-ordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, Kuala Lumpur, 3Medical Scientist Training Program, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045,

4Department of Oral and Maxillofacial Surgery, Hospital Kuala Lumpur, Malaysia, 5Department of Oral & Maxillofacial Surgery, Tengku

Ampuan Rahimah Hospital, Klang, Malaysia, 6Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Malaya, Malaysia.

ABSTRACT

Cancer cell lines are essential tools in the understanding of tumour biology. Further, these in vitro models

are important screening tools for the identification of anti-cancer compounds. Recent high-throughput

genetic analysis of cell lines show that tumour heterogeneity commonly observed in cancers can be

recapitulated in large panel of cancer cell lines. More importantly, gene expression and mutational

analysis of cell lines that correspond to drug response affords an opportunity to identify biomarkers that

correlate specifically with therapeutic outcomes. We have established one of the largest panel of Asian

oral cancer cell lines and characterized these extensively for growth characteristics and molecular

alterations. More importantly, these cell lines are genetically representative of the tumours from which

they were derived making them ideal models to study oral cancer development. Common genetic

mutations that were observed in the cell lines were TP53 mutations (11/16), CDKN2A alterations (8/16)

and NOTCH mutations (8/16) amongst others. Notably, the OSCC cell lines could be sub-classified into 3

sub-groups based on their enriched pathways. Based on the genetic profiles of these cell lines, we have

identified several drugs that could be efficacious in OSCC and tested several of these in vitro. In

particular, we demonstrated that the majority of OSCC cell lines were exquisitely sensitive to Dasatinib, a

multiple kinase inhibitor. The use of in vitro models affords an opportunity to study the mechanism by

which Dasatinib acts on OSCC cell lines and could be used to further refine the biomarkers that are

associated with drug response.





Inhibition of the Interferon Inducible Transmembrane Protein (IFITM) Suppresses

Cell Proliferation by Inducing Cellular Senescence

1KK Sam ,

1 CP Gan,

2 WM Wan Mustafa,

3SM Ismail,

4 RB Zain and

1,4SC Cheong

1Oral Cancer Research Team, Cancer Research Initiatives Foundations, Sime Darby Medical, Subang Jaya, Malaysia, 2Dept of Oral &

Maxillofacial Surgery, Hospital Kuala Lumpur, Malaysia, 3Dept of Oral & Maxillofacial Surgery, Faculty of Dentistry, University Malaya,

Kuala Lumpur, Malaysia, 4Oral Cancer Research and Coordinating Centre, University Malaya, Kuala Lumpur, Malaysia

ABSTRACT

Oral cancer is one of the top ten cancers worldwide. Despite the advancements in cancer therapy, the 3-

year survival rate among oral cancer patients remains poor at 50 %. Interferons play a critical role in

tumour pathogenesis by controlling cell proliferation, differentiation and apoptosis and Interferon

inducible transmembrane protein (IFITM) family of proteins have several functions including cell

adhesion, anti-proliferation in addition to its anti-viral activity. Notably, IFITM genes have been found to

be deregulated in various types of solid tumours. IFITM3 was previously found to be up-regulated in 80

% of oral squamous cell carcinoma (OSCC) and in this study, we investigated the role of IFITM3 in oral

cancer pathogenesis. The expression of IFITM3 in two OSCC cell lines, ORL 150 and ORL 204 was

knockdown by si-RNA and the role of IFITM3 in driving cell proliferation, migration and invasion was

investigated. We found that the down-regulation of IFITM3 significantly inhibited cell proliferation,

migration and invasion suggesting that IFITM3 is a potent driver of OSCC. Morphologically, cells that

had reduced or loss of IFITM3 appeared flattened, had increased nucleoli and moreover expressed -

galactosidase which are characteristics of senescent cells. This data suggest that IFITM3 confers a

survival advantage to OSCC cells and targeting this gene could result in tumour growth inhibition by the

induction of senescence.

P39





Identification and Evaluation of FJX1-Derived Peptide Vaccine Candidates for

Nasopharyngeal Carcinoma Patients

* 1S.J. Chai,

2Y.Y. Yap,

3Y.C. Foo,

1, 4L.F. Yap,

5S. Ponniah,

1S.H. Teo,

1K.P. Lim

1 Cancer Research Initiatives Foundation, Subang Jaya, Malaysia, 2 Department of Surgery, Clinical Campus Faculty of Medicine and Health

Sciences, Universiti Putra Malaysia, Hospital Kuala Lumpur, Malaysia, 3 Department of Oncology, Sime Darby Medical Centre, Subang Jaya,

Malaysia, 4 Dental Research & Training Unit and Oral Cancer Research & Coordinating Centre, Faculty of Dentistry, University of Malaya,

Kuala Lumpur, Malaysia, 5 Cancer Vaccine Development Program, Department of Surgery, Uniformed Services University of the Health

Sciences, Bethesda, USA.

ABSTRACT

Nasopharyngeal carcinoma (NPC) is the fourth most common cancer among Malaysians. Although the

majority of patients respond well to concurrent chemoradiotherapy, distant metastases and recurrence

remain the major cause of death. In our previous study, we identified four-jointed box one (FJX1) as a

tumour antigen and potential therapeutic target. In this study, we explored the efficacy and specificity of

FJX1-derived peptides as candidate peptide vaccine to elicit T cells response in donors and NPC patients.

Six MHC class I and 2 MHC class II-restricted FJX1 peptides were generated and screened for the

immunogenicity, cytolytic response and specificity towards cancer cells using ELISPOT assay. Our

results showed that these peptides are immunogenic and T cells from NPC patients can be stimulated to

display peptide-specific cytolytic activity. Out of the 8 peptides tested, 4 exhibited higher killing ability

and specificity against FJX1-expressing NPC cells compared to control cells, suggesting that these

peptides are capable of inducing specific anti-tumour response. In conclusion, these FJX1-derived

peptides could be potential target to treat NPC and recurrences of NPC. Nonetheless, further investigation

is required to determine the toxicity and the usefulness of these peptides.

P40





Metformin Synergize 5-Fluorouracil, Epirubicin and Cyclophosphamide (FEC)

Sensitivity in Breast Cancer Stem Cells and Non-Stem Cancer Cells

*

1,2S.S. Soo,

3B. S. Tan,

2C.H. Ng,

2N. A. Mohd Taib,

4C. H. Yip,

1,2S. H. Teo,

3C. O. Leong

1 Cancer Research Initiatives Foundation, Selangor, Malaysia, 2 University Malaya Cancer Research Institute, University of Malaya, Kuala

Lumpur, Malaysia, 3 International Medical University, Kuala Lumpur, Malaysia, 4 Sime Darby Medical Centre, Selangor, Malaysia

ABSTRACT

Tumorigenic cancer stem cells (CSCs) existing within a population of tumor cells are responsible for

therapeutic resistance and may be the underlying cause for cancer progression, recurrence and metastasis.

Recent evidence suggests a link between AMPK-mTOR and cancer cell survival. An anti-diabetic,

AMPK-activating drug, metformin has been recently shown to reduce cancer incidence and cancer related

death. Most importantly, metformin is able to selectively kill CSCs and acts together with chemotherapy

to block tumor growth and prolong tumor remission. However, it is unknown whether other

pharmaceutical AMPK/mTOR-acting drugs will elicit similar effects as metformin. Thus, we seek to look

at the combination effect of AMPK/mTOR-acting drugs and chemotherapy on breast CSCs and non-stem

cancer cells. The anti-cancer effect of metformin, AICAR, A-769662 and rapamycin in combination with

cisplatin, doxorubicin, paclitaxel and FEC chemotherapies on breast cancer cell lines were investigated.

Mammospheres derived from attached non-stem cancer cell lines were used to study CSCs.

CellTiter96®Aqueous One Solution was utilized to measure cell proliferation. Compound C (AMPK-

inhibiting drug) and transient AMPK shRNA knockdown was utilized to investigate AMPK dependency

of the treatments. Mammospheres had higher IC50 when treated with chemotherapy drugs and

AMPK/mTOR-acting drugs except for metformin compared to attached cells. Combination of metformin

and FEC chemotherapy exhibited synergistic effect on both mammospheres and attached cells. Inhibition

of AMPK completely abrogated the anti-tumor effects of metformin or its synergistic effects with FEC on

non-stem cells. However, inhibition of AMPK has no effect on metformin sensitivity on mammospheres.

Taken together, our results demonstrate that CSCs are more sensitive to metformin. Combination of FEC

with metformin achieved a synergistic effect. The effect of metformin is AMPK-independent in CSCs

although dependent in non-stem cells. Thus, metformin still remains a potential therapy in targeting

cancer stem cells.

P41





Trifluoromethoxy-Goniothalamin Induces Caspase-2-Mediated Chronic Myeloid

Leukemia K562 Cells Apoptosis through Early Bcr-Abl Dephosphorylation and

p38 MAPk Activation

1KM Chan,

2, * KL Pang,

3JFF Weber and

4SH

Inayat-Hussain

1 Environmental Heath Programme, School of Diagnostic Science & Applied Health, Faculty of Health Sciences, Universiti Kebangsaan

Malaysia, Malaysia, 2 Biomedical Science Programme, School of Diagnostic Science & Applied Health, Faculty of Health Sciences, Universiti

Kebangsaan Malaysia, Malaysia, 3 Institute Of Natural Products For Drug Discovery, Faculty of Pharmacy, Universiti Teknologi Mara, Malaysia.4 Petroliam Nasional Berhad (PETRONAS), PETRONAS KL Twin Tower, Malaysia.

ABSTRACT

Chronic myelogenous leukaemia (CML) is a hematological stem cells cancer caused by a chromosomal

translocation t(9;22)(q34;q11). The chimeric protein, Bcr–Abl is generated with constitutively tyrosine

kinase activity and subsequently activates several downstream signal effectors and signaling pathways.

Styryllactones, secondary metabolites of Goniothalamus plant are served as potential chemotherapeutic

drug candidate. Previous studies demonstrated that styryllactone selectively induce cytotoxicity primarily

apoptosis in several cancerous cells. Recently a novel styryllactone was synthesized namely Z-6-[2-(4-

trifluoromethoxy-phenyl)-vinyl] -5,6-dihydropyran-2-one or in short trifluoromethoxy-goniothalamin

(TFGN), whereby its biological activity has not been well characterized. In this study, the molecular

mechanism of apoptosis induced by TFGN was investigated in K562 chronic myeloid leukemia cells

which harbor high level of Bcr-Abl. Our results demonstrate that 24h TFGN treatment induced apoptosis

in K562 cells in a concentration-dependent manner based on phosphatidylserine externalization.

Interestingly, an early simultaneous phosphorylation of p38 MAPK and dephosphorylation of Bcr-Abl

kinase and it’s substrate, CrkL were seen in TFGN-treated K562 cells as early as 15 minutes. This is the

first demonstration of the role of p38 MAPk and Bcr-Abl inhibition in stryryllactone-induced apoptosis.

The cells progressed to apoptosis with early activation of caspase-2 at 1 hour of TFGN treatment and lead

to activation of caspase-9. There was a loss of mitochondrial membrane potential and the processing of

executioner caspase-3 in later stage. We further confirmed the role of caspase-2 as the initiator caspase

whereby pretreatment with specific caspase-2 inhibitor (z-VDVAD-FMK) protects K562 cells from

TFGN-induced apoptosis. In conclusion, TFGN induced caspase-2-mediated apoptosis through Bcr-Abl

inhibition and early p38 MAPk phosphorylation in K562 cells. These data provide insights for the

development of styryllactones specifically TFGN as a potential therapeutic agent for chronic myeloid

leukemia.

P42





TMPRSS2-ERG Translocation Status in A Multi-Ethnic Cohort of

Prostate Cancer Patients from Malaysia.

1Gregory M. Kelly,

1Yink Heay Kong,

2Rajadurai Pathmanathan,

3,4Tan Hui Meng,

5Tan Shyh Han and

1Cheong Sok

Ching.


Jaya, Selangor Darul Ehsan, Malaysia 2Department of Pathology, Sime Darby Medical Centre,47500 Subang Jaya, Selangor, Malaysia. 3Medical Research Development Unit, University of Malaya, Kuala Lumpur, Malaysia. 4Department of Urology, Sime Darby Medical Centre, Kuala

Lumpur, Malaysia. 5Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences,

Bethesda MD, USA.

ABSTRACT

Prostate cancer prevalence as well as the genomic alterations associated with this disease has been shown

to vary in different geographical locations in the world. The most frequent genetic event that occurs in

prostate cancer is a gene fusion between the regulatory sequence of the androgen receptor responsive

genes (predominantly TMPRSS2) and the protein coding sequence of the nuclear transcription factor of

the ETS gene family (predominately ERG). This TMPRSS2-ERG gene fusion results in the over-

expression of a truncated ERG protein and is highly prevalent in prostate cancer patients from Western

countries. The prevalence of this gene fusion is not well-studied in Asian populations. 175 formalin-fixed,

paraffin-embedded specimens from 107 patients diagnosed at the Sime Darby Medical Centre, Subang

Jaya, Malaysia were included in this study. The sections were analyzed for the TMPRSS2-ERG fusion

event by looking at ERG expression by immunohistochemistry. Of the 175 sections that were stained,

positive ERG expression was found in 34 of 135 (25.2%) Chinese patients, 1 of 14 (7.1%) Malay patients,

12 of 15 (80%) Indians patients and 3 of 11 from other ethnicities. The overall frequency of the

TMPRSS2-ERG fusion event, which was detected using a monoclonal antibody in the tested Malaysian

population, was 28.6%. The overall frequency of ERG over-expression from the Malaysian population is

consistent with frequencies that have been observed in other Asian countries such as Japan, Korea, China

and India. This observed ERG frequency is much lower than those recorded in Europe and America.

Further evaluation into the occurence of this genetic event is needed particularly in the Indian and Malay

populations to determine the true frequency of this genetic event and its implications in Malaysian

prostate cancer patients.

P43





In Vitro Cytotoxicity of Strobilanthus crispus Ethanol Extract on

Hormone Dependent Human Breast Adenocarcinoma MCF-7 Cell

1Chong HZ,

1*Asmah R,

2Abdah MA,

3Norjahan Banu MA,

4Gwendoline Ee CL,

2Fauziah O,

3 Yeap SK

1Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences; 2Department of Biomedical Science, Faculty of Medicine and

Health Sciences; 3Department of Cell Biology and Molecule, Faculty of Biotechnology and Biomolecular Sciences; 4Department of Chemistry, Faculty of Science,Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

ABSTRACT

Strobilanthuscrispus has been traditionally used as antidiabetic, anticancer, diuretic, antilytic and laxative

agent. However, cytotoxicity and antiproliferative effect of S.crispus is still

unclear.Strobilanthuscrispuswas able to reduce cell viability and proliferation in MTT and BrdU assays.

Both cell cycle progression and Tunnel assay suggested that IC50 of S.crispusethanol extract induced sub-

GI cell cyclephase, and DNA fragmentation. On the other hand, translocation of mitochondria

cytochrome c release, induction of caspase 3/7 and p53 while suppress XIAP on treated MCF-7 cell were

also observed in this study. Our findings suggest that S.crispusethanol extract induced apoptosis and

DNA fragmentation on hormone dependent breast cancer cell line MCF-7 via mitochondria dependent

p53 apoptosis pathway.

P44





Silencing of PROS1 Induces Apoptosis and Inhibits Migration and Invasion of

Glioblastoma Multiforme Cells

1Mohd Firdaus Che Mat,

1Kamariah Ibrahim,

1Nor Azian Abdul Murad ,


2Nuraziah

Mohammad Zain, 3Nurismah Md Isa,

1A Rahman A Jamal and

1Roslan Harun

1 UKM Medical Molecular Biology Institute (UMBI), 2Faculty of Health Sciences UKM, 3Faculty of Medicine, UKM Medical Center,

Kuala Lumpur

ABSTRACT

Protein S (alpha) (PROS1)is a cofactor for the anticoagulant protease, activated protein C (APC) that

functions to inhibit blood coagulation. Meta-analysis of five microarray gene expression datasets also

showed that PROS1 was highly expressed in glioblastomamultiforme (GBM). However, the mechanisms

how PROS1 contributes to cancer development and progression are still not known. In this study we

determined the effects of PROS1 gene silencing on various cellular functions that were important in

cancer. Synthetic lethal RNA interference (SMARTpoolTM

, Dharmacon) was used to knockdown PROS1

expression in human glioblastomacell line (PTEN-positive), LN-18. The percentage of PROS1

knockdown was measured at 24 and 48hours using qPCR and western blot. The effects of PROS1

knockdown on the phenotypic changes including cell viability, migration, invasion, apoptosis, and cell

cycle wereevaluated using the standard methods.Silencing of PROS1 resulted in 81.6% and 59.1%

reduction in the cell viability at 24 and 48 hours respectively. The reduction in cell viability was caused

by the increase in apoptosis using ssDNA Apoptosis ELISA kit. There was no effect of PROS1 inhibition

on cell cycle. Interestingly, PROS1 knockdown resulted in significant reduction inLN18 cell migration

and invasion.In conclusion, PROS1 may have important roles in GBM progression. Targeting PROS1

might be a potential therapeutic strategy for GBM.





Determining microRNAs Profile in Formalin-Fixed Paraffin-Embedded Samples

of Metastatic Serous Ovarian Cancer

1Fateen Farhana Ibrahim

*,

1Saiful Effendi Syafruddin,

3Siti Aishah MdAli,

3Reena MdZin,

1Rahman Jamal,

1,2Norfilza Mohd Mokhtar.

1UKM Medical Molecular Biology Institute, 2Department of Physiology, 3Department of Pathology, Faculty of Medicine,

Universiti Kebangsaan Malaysia.

ABSTRACT

Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy worldwide with the

serous is the commonest subtype. Patients are often diagnosed at the late-stage and eventually succumb to

this metastatic disease. A small endogenous non-coding molecule known as microRNA (miRNA) has

emerged to play critical roles in orchestrating metastasis process. Unfortunately, the knowledge on

miRNA-regulating metastatic genes in serous EOC is still lacking. Hence, we aimed to identify the

potential microRNAs that regulate the metastatic genes in FFPE of serous EOC. We extracted miRNA-

enriched from twenty-two serous ovarian cancer patients with metastatic evidence (Stage II–IV) and

twenty-two unmatched non-cancerous ovaries using High Pure miRNA Isolation Kit. We conducted

miRNA expression profiles on Cancer Focus microRNA PCR Panel using SYBR Green Master Mix and

analyses were performed using the GenEx software. A total of 36 miRNAs were differentially expressed

in serous ovarian cancer compared with normal samples (p-value<0.05, fold-change≥2.0). Out of these,

19 were up- and 17 were down-regulated. Six up-regulated miRNAs namely miR-200c-3p, miR-200b-3p,

miR-200a-3p, miR-141-3p, miR-182-5p and miR-21-5p, were either reported in serous ovarian cancer

studies or other cancers. Most notably, we identified two novel up-regulated miRNAs, miR-7-5p and

miR-31-5p that were predicted to be linked to the metastatic capacity of the cancer. We validated these

miRNAs individually using qRT-PCR and the results obtained did confirm their aberrant expressions. In

conclusion, the differentially expressed miRNAs could serve as promising biomarkers in detecting

metastatic serous ovarian cancer as well for future therapeutic target. Further analysis on their localisation

in clinical samples and functional analysis through in-vitro studies are required to show their significant

involvement in the metastatic serous EOC.

P46




8-10thNovember 2013, The Royale Chulan,Kuala Lumpur

Identification of Genetic Alterations in Dukes’ B and C Colorectal Cancers

1Shafina Nadiawati Abdul,

1Nurul Syakima Ab Mutalib,

3Ismail Sagap,

4Isa Mohamed Rose,

1,5Roslan Harun,

1A

Rahman A Jamal and 1,2

Norfilza Mohd Mokhtar

1UKM Medical Molecular Biology Institute, 2Department of Physiology, Faculty of Medicine, 3Department of Surgery, Faculty of Medicine,

4Department of Pathology, Faculty of Medicine,5Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur,

Malaysia

ABSTRACT

Colorectal cancer (CRC) is one of the major causes of deaths worldwide. Staging of CRC is challenging

due to inconsistent views on the current staging system. Classifying CRC into Dukes’ staging seems to be

the most challenging issue. As a result, the effective treatment for this cancer is hard to implement. Next

generation sequencing will enable identification of mutations underlying these events and may contribute

to discovery of new biomarkers in CRCs. The objective of this study was to identify genetic mutations in

Dukes’ B and C of CRC patients. Genomic DNA was extracted from fresh tissues of Dukes’ B (n=4) and

Dukes’ C (n=1) and subjected to sequencing using Ion Ampliseq Comprehensive Cancer Panel. Two

deleterious mutations were commonly identified namely PDE4DIP p.R25L and ITGB2 p.Q354H in

Dukes's B CRC as compared to normal samples. Deleterious mutations in ten genes were identified in

Dukes’ C in which 8 out of 10 were unique for this stage. These included TRIP11 p.G1827S and TP53

p.R175H; p.R175P; p.175L. There were also novel mutations namely PER1 p.G1066V, GF2 p.H1400,

ITGA9 p.V323L, NOTCH4 p.C2645, UBR5 p.H757p, MAPK2K1 p.L375p, IRS2 p.S894Y, SMUG1

p.P122L. The mutation involving the E3 ubiquitin-protein ligase (UBR5 p.H757P; histidine to proline

mutation) was a novel missense mutation resulting in altered UBR5 protein function. This gene is

localised to chromosome 8.q22 and is involved in ubiquitin mediated proteolysis pathway that plays an

essential function in regulation of cell cycle, control of signal transduction pathways, development and

differentiation. Integrin alpha 9 (ITGA9 p.V323L) was also a novel deleterious mutation that was unique

to our samples. It could regulate diverse biological functions such as angiogenesis, lymphangiogenesis,

cancer cell proliferation and migration. This preliminary finding displayed the value of Ion Torrent PGM

for the rapid screening of mutations and we hope that on completion of the profiling in all the samples, we

are able to shed light on molecular characterisation of Dukes’ B and C of CRC.





Identifying Differentially Expressed Genes from RNA Sequencing Data of

Azoxymethane Induced Aberrant Crypt Foci in F344 rat

1Mohammad Khusni Ahmat Amin,

2Yasmin Anum Mohd Yusof,

1A Rahman A Jamal,

1Wan Zurinah Wan Ngah

1UKM Medical Molecular Biology Institute, Kuala Lumpur, 2Department of Biochemistry, Faculty of Medicine, Jalan Raja Muda Abdul Aziz,

50300, Kuala Lumpur, Malaysia

ABSTRACT

The mechanism of Aberrant Crypt Foci’s (ACF) progression to colon cancer is not well elucidated. Thus,

high-throughput sequencing enables expression analysis at the level of individual transcripts. The aim of

this study was to determine the differentially expressed genes (DEG) in Azoxymethane (AOM) induced

ACF by using RNA sequencing. A total of 18 Male F344 rats were divided into 2 groups: normal + olive

oil (vehicle) and AOM + olive oil. ACF was induced by subcutaneous injection of AOM. After 8 weeks,

the RNA samples were extracted from colon and RNA sequencing was performed. The sequencing data

were aligned and mapped to Rattus norvegicus reference genome by CLC Genomics Workbench

Software. Further analysis was done by DAVID Bioinformatics’ tool. The sequencing data were mapped

to 26313 genes with an average of 17.3M reads. The induction of ACF by AOM compared to vehicle

group resulted in 414 genes (241 ↑, 173 ↓) at the proximal colon, 293 genes (203 ↑, 90 ↓) at the middle

colon,419 genes (218 ↑, 201 ↓) at the distal colon. Similar activities were identified in the proximal and

middle colon where most of DEG were involved in translational elongation, cytoplasmic vesicles and

ribosomal activity. Meanwhile, the process of neuron system and cell development were significantly

identified in the distal colon. Two common pathways in cancer, namely Ribosome and O-Glycan

biosynthesis were identified in all regions. Interestingly, Focal adhesion pathway was only identified in

the proximal and middle colon. In addition, ECM receptor interaction was found in the proximal colon,

glutathione metabolism in the middle colon and fructose and mannose metabolism in the distal colon.

Results showed that the formation of ACF may involve among the identified KEGG pathways based on

the significant DEG and deserve further investigation to look for potential biomarkers.





Characterisation of microRNA as the Biomarkers for Metastatic Serous

Ovarian Cancer

2Zahreena Othman,

3Ahmad Zailani Hatta Mohd Dali,

3Yulianty Arifuddin,

4Siti Aishah Md Ali,

4Reena Rahayu Md

Zin, 1A

Rahman A Jamal and


1UKM Medical Molecular Biology Institute, 2Department of Physiology, 3Department of Obstetrics & Gynaecology, 4Department of Pathology,

Faculty of Medicine, Universiti Kebangsaan Malaysia

ABSTRACT

It is estimated more than 200 000 women develop ovarian cancer and approximately 100 000 die

from the disease every year. Serous ovarian adenocarcinoma is the commonest subtype which

accounts for nearly 50% of all ovarian cancers. This type of cancer is usually asymptomatic in

the early stages and often diagnosed at late stages when the cancer has metastasised to distant

sites. Thus, hindering the possibility of providing curative treatments. Little is known regarding

the roles of small, non-coding molecules called microRNA (miRNA) in the regulation of genes

related to metastatic serous ovarian cancers. The purpose of the present work was to investigate

and address this issue. A total of twenty-three snap-frozen samples were used in this study which

consist of eleven serous ovarian cancer (Stage II - Stage IV) and twelve, unmatched normal

samples. Only samples with more than 80% cancer cells were used and total RNA was extracted

using QiagenmiRNeasy Mini Kit. RNA integrity number of more than 6 was used. MiRNA

expression profiling was performed on miRNAqPCR Cancer Focus Panel using SYBR Green

real-time PCR. qPCR results were then analysed using Genex software. Unpaired t-test revealed

10 and 16 miRNAs to be up-regulated and down-regulated respectively in tumour compared to

normal samples (p < 0.05 with Bonferroni correction). Among the up-regulated miRNAs were

miR-106a, miR-18a, miR-203, miR-93, miR-141 and miR-7. The up-regulation of miR-141,

miR-18a and miR-93 in ovarian cancer were found to be significantly correlated with poor

prognosis. As a conclusion, current results identified several miRNAs which may have important

roles in the regulation of gene expression in metastatic serous ovarian cancer. These miRNAs

may also serve as potential biomarkers for early detection of the disease, which eventually may

offer possible therapeutic treatments to patients.





Gamma-tocotrienol Treatment Increased Peroxiredoxin-4 Expression in HepG2

Liver Cancer Cell Line

1Zakiah Jubri,

1Farahani Abdul Rahman Sazli,

2Mariati Abdul Rahman,

3Saiful Anuar Karsani,

4Abdul Gapor Md

Top, 1Wan Zurinah Wan Ngah

1Department of Biochemistry, Faculty of Medicine, 2Department of Clinical Oral Biology, Faculty of Dentistry, Universiti Kebangsaan

Malaysia, 3Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur and University of Malaya centre for

Proteomics Research (UMCPR), 4Malaysian Palm Oil Board, Bangi, Selangor, Malaysia

ABSTRACT Gamma-tocotrienol (GTT) is a member of the vitamin E family and has been reported to have

antiproliferative effect against a wide variety of cancer cells. Several possible mechanisms have been

proposed to explain the antiproliferative activity of GTT and the protein involved. But up to now the

pathway is still not yet confirmed. This study was to determine the antiproliferative effect of gamma-

tocotrienol (GTT) treatment on differential protein expression in HepG2 cell. HepG2 cells was treated

with 70 µM GTT for 48 hours and differentially expressed protein spots were determined by two-

dimensional electrophoresis (2DE), identified by MALDI-TOF mass spectrometer (MS) and validated by

quantitative real-time polymerase chain reaction (qRT-PCR). GTT treatment on HepG2 cells showed a

total of five differentially expressed proteins when compared to their respective untreated cells where

three proteins were down-regulated and two proteins were up-regulated. One of these upregulated

proteins was identified as peroxiredoxin-4 (Prx4). Validation by qRT-PCR however showed decreased

expression of Prx4 mRNA in HepG2 cells following GTT treatment. GTT might directly influence the

expression dynamics of peroxiredoxin-4 to control proliferation in liver cancer.





Serum Proteomics-based Identification of Differentially Expressed Proteins in

Colorectal Cancer

1Lay Cheng Lim*,

2Mee Lee Looi,

3Ismail Sagap,

4Isa Mohammed Rose,

1Syed Zulkifli Syed Zakaria,

1Rahman Jamal

1UKM Medical Molecular Biology Institute, 3Department of Surgery, 4Department of Pathology, Universiti Kebangsaan Malaysia Medical

Center, Universiti Kebangsaan Malaysia; 2School of Biosciences, Taylor’s University Lakeside Campus.

ABSTRACT The high incidence and mortality rate of colorectal cancer (CRC) showed the inefficiency of CRC

screening procedures at present. Besides, serum tumour markers such as carcinoembryonic antigen (CEA)

and CA19-9 are limited by its poor sensitivity and specificity. Hence to identify the potential blood-based

protein biomarkers in CRC, we compared serum protein profiles from ten early stage CRC (Dukes’ A and

Dukes’ B), ten late stage CRC (Dukes’ C and Dukes’ D) and ten normal controls. Multiple affinity

removal system spin cartridge (MARS) was use to deplete fourteen high abundance proteins present in

the serum samples prior to tandem mass spectrometer run. Progenesis software analysis showed twenty

two proteins were significantly differentially expressed between groups (p<0.05). It was found seventeen

proteins were upregulated and five proteins were downregulated in CRC as compared to normal controls.

These proteins were identified to play key roles in the acute phase and cellular defense response,

proteolysis, complement pathway, blood coagulation, immunity, transport system and regulation of

phosphatase activity. Further verification of these proteins is ongoing with the hope to find characteristic

patterns of protein biomarkers expression in CRC.





Comparing Supplementation of Palm Tocotrienol-Rich Fraction with Alpha-

Tocopherol in Human: A Preliminary Study

*Siti Madiani Abdul Ghani, Goon Jo Aan and Wan Zurinah Wan Ngah

Department of Biochemisty, Faculty of Medicine, Universiti Kebangsaan Malaysia Kuala Lumpur, Malaysia

ABSTRACT

The role of free radicals in age-related diseases aroused the interest on using vitamin E, the most

important lipid-soluble antioxidant, for disease chemoprevention and delaying aging process probably via

ROS scavenger mechanism and modulation of gene expression. Most vitamin E supplements available in

market however only contain α-tocopherol which is only one out of eight natural forms of vitamin E.

Studies that reported natural α-tocopherol supplementation significantly depressed bioavailability of α-

tocotrienol suggested the use of tocotrienols containing minimum amount of α-tocopherols such as

tocotrienol-rich fraction (TRF) instead of α-tocopherol alone. In this study, the differential effect of palm

TRF and α-tocopherol isomers were compared. Thirty six healthy volunteers aged 50-55 years old were

recruited and randomly divided into three groups supplementation consist of Tri.E tocotrienol (150 mg/d),

α-tocopherol (400 IU/d) or identical placebo (control). Fasting blood (30 ml) was taken at baseline

(month 0), 3 months and 6 months of supplementation for measure the general health which includes

haematological profile, liver function test, lipid profile, renal profile and fasting blood glucose (FBG) and

oxidative stress markers such as DNA damage. Our preliminary analysis showed that Tri.E tocotrienol

supplementation for 6 months significantly decreased the total DNA damage (p<0.05) compared to

control. In conclusion, palm Tri.E tocotrienol supplementation may have a greater effect in preventing

DNA damage compared to α-tocopherol.





Gene Expression Changes in Azoxymethane-Induced Colon Cancer in Fischer 344

Rat and Modulation by Piper betle Aqueous Extracts

1Azleen Mat Sharif*,

2Yasmin Anum Mohd Yusoff,

1Mohammad Khusni Ahmat Amin,

1Abdul Rahman A Jamal,


1UKM Medical Molecular Biology, PPUKM and 2Department of Biochemistry, Faculty of Medicine, UKM

ABSTRACT

Studies on the anti-tumorigenesis of P. betle (PB) in various types of tumors have been widely reported

but is still limited in colon cancer. In this study the gene expression in normal and azoxymethane (AOM)-

induced colon cancer in rat modulated by PB extracts was determined. Total RNA from rat colon tissues

were extracted, purified and converted to cDNA using TruSeq RNA Sample Preparation kit based on the

sample preparation protocol provided by the manufacturer (Illumina, San Diego, CA). Quality control and

differentially expressed genes (DEGs) identification among samples were performed by using CLC

Genomic Workbench software version 5.5.1. We detected total 3 (2 ↑, 1 ↓), 421 (143 ↑, 278 ↓) and 868

(712 ↑, 156 ↓) significant DEGs between PB, AOM and both AOM and PB at 8 weeks treatment

respectively. The overlapping of DEGs detected among the three colon regions (proximal, middle and

distal) for AOM and PB treatment was performed using GeneVenn diagram tools. Ten DEGs were

ENSRNOG00000004203 (↓), BACE2 (↓), EID1 (↓), HEPACAM2 (↓), OCLN (↓), SFRP1 (↑) GADD45B

(↑), SOCS3 (↑), RETNLG (↑) and HAS1 (↑) while 9 DEGs all were upregulated such as ITGA6,

GALNT7, ADAMDEC1, C4A, BCAS1, C1GALT1, ENSRNOG00000048902 and COL6A5 were common

to all three colon regions for both AOM and PB treatment respectively. Functional enrichment analysis of

differentially expressed genes were conducted using online tools from DAVID and were detected in

several commonly enriched KEGG pathways with DEGs list in AOM and PB treatment. Among detected

enriched KEGG pathways, O-Glycan biosynthesis pathway was commonly affected in both proximal and

middle part of colon for AOM and PB treatment groups. PB extracts caused changes in genes expression

when given in both normal and AOM-induced colon cancer in rat. This gene-level summarization

approach also provides the basis for further molecular understanding of PB bioactivity at the functional

level as well as in identifying biomarkers.





Differential Protein Profiles in Meningioma And Glioma: A Preliminary Glimpse

1 MR Zam Zureena,

1SF Chin,

1,2BH Soon,

2A Abu Bakar,

2CJ Toh,

2F Fadzil,

2J Thanabalan,

3MS Mohd Haspani,

2R Kumar,

1H Roslan,

1WZ Wan Ngah,

1NM Mokhtar,

1R Jamal

1UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Malaysia, 2Division of Neurosurgery, Department of Surgery,

Faculty of Medicine, Universiti Kebangsaan Malaysia, Malaysia, 3Neurosurgery Department, Hospital Kuala Lumpur, Malaysia.

ABSTRACT

The identification of type- and grade-specific protein biomarkers for brain tumors will provide the basis for better

diagnosis, prognosis and tailored treatments for the patients. To date, glioma and meningioma were classified based

on morphologic characteristics according to the WHO classfication. The aims of this study were to identify the

differentially expressed proteins between glioma and meningioma, and to trace the proteome pattern corresponding

to the WHO cancer grading. In this preliminary study, 15 glioma and 12 meningioma tissues of different grades

were obtained from patients diagnosed with primary brain tumours who underwent surgical excision at either the

UKM Medical Centre or the Kuala Lumpur Hospital. The proteins were extracted, digested and analyzed on a liquid

chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS). Differentially

expressed proteins were identified and classified by using the Progenesis LCMS and Protein Analysis Through

Evolutionary Relationship (PANTHER) softwares, respectively. A total of 50 proteins were identified in glioma, in

which 21 proteins were overexpressed in low grade and 29 proteins in high grade glioma. As for meningioma, 21

differentially expressed proteins were identified with 9 proteins from low grade and 12 proteins in high grade tumor.

Overall, the differentially expressed proteins in glioma and meningioma were predominantly proteins involved in

structural molecules (Glioma: Glial fibrillary acidic protein, Tubulin beta-4A chain, Tubulin alpha-1C chain,

Tubulin beta-2A chain and Tubulin beta-6 chain; Meningioma: Vimentin, collagen alpha-1 (I) chain and Chloride

intracellular channel protein 1) and catalytic activities (Glioma: Pyruvate kinase isoenzymes M1/M2, Isocitrate

dehydrogenase (NAD) subunit beta mitochodrial, Dihydropyrimidinase-related protein 3, Glyceraldehyde-3-

phosphate dehydrogenase, Creatine kinase B-type and Fructose bisphosphate aldolase C; Meningioma: Chloride

intracellular channel protein 1, Transketolase, L-lactase dehydrogenase A chain, Protein disulfide-isomerase A3 and

Aldehyde dehydrogenase, mitochondrial). In addition, we observed a differential protein pattern in glioma and

meningioma in which most of the identified upregulated proteins in gliomas involved in binding and transporter

activity were from high grade samples whereas for meningioma, the identified upregulated proteins involved in

binding, transporter, translation regulator, receptor and ion channel activities were from low grade samples. We have

identified differential proteomic profiles that may characterize and distinguish glioma and meningioma in relation to

the tumour grade. Further validation of these proteins by immunohistochemistry is underway.

P54




8-10thNovember 2013, The Royale Chulan, Kuala Lumpur

Keratoconus and VSX1 Polymorphism: A Preliminary Study in

A Malaysian Population

1*

JB Ng, 1RYY Poh,

1SM Noor,

1JAMA Tan,

2V Subrayan,

3JP Deva

1 Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia, 2 Department of

Ophthalmology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia, 3 Faculty of Medicine and Health Sciences,

Department of Surgery, Universiti Tunku Abdul Rahman, Selangor, Malaysia.

ABSTRACT

Keratoconus is a progressive disorder characterised by thinning of the cornea, and is associated with

consanguinity, ultraviolet radiation, eye-rubbing and allergy. Visual system homeobox I (VSX1) gene is

expressed in the human cornea and has been controversially implicated in keratoconus, wherein it was

pathogenic in some populations while being a variation in gene frequency in the others. Molecular genetic

analysis was conducted on 50 keratoconus patients and 100 unrelated controls. DNA extracted from

EDTA blood samples were amplified via polymerase chain reaction (PCR) for the VSX1 polymorphisms

at exons 2 and 4, and resulting PCR products were sequenced. The nucleotide sequences were aligned

with the published VSX1 cDNA sequence (Genbank accession number NM_014588) using Sequencher

5.1 (Gene Codes Corporation). Polymorphisms in exon 2 of VSX1 were seen in two of the controls but not

in keratoconus patients. The absence of mutation in keratoconus suggests that this is a polymorphism

within the study’s population. Similar findings were reported in Italian, Slovenian and Saudi Arabian

populations. Conversely, VSX1 has been reported to be pathogenic in other diverse populations such as in

the Iranians, Indians, Canadians and Koreans. VSX1 gene with reference to exons 2 and 4 may not play a

major role in the pathogenesis of keratoconus in the Malaysian population. Sample collection and

genotyping is currently ongoing.

P55




8-10thNovember 2013, The Royale Chulan, Kuala Lumpur

Identification of Genetic Variants in Molecular Subtypes of Endometrioid

Endometrial Adenocarcinoma Using the Ampliseq™ Comprehensive Cancer Panel

1Siti Syazani Suhaimi,

1Hanaa’ Abdul Rahman,


3Ahmad Zailani Hatta Mohd Dali,

1,5Roslan Harun,

4Reena Rahayu Md Zin,

1A Rahman A Jamal and


1UKM Medical Molecular Biology Institute, 2Department of Physiology, Faculty of Medicine, 3Department of Obstetrics and Gynaecology,

Faculty of Medicine, 4Department of Pathology, Faculty of Medicine,5Department of Medicine, Faculty of Medicine,

Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

ABSTRACT

Endometrioid endometrial adenocarcinoma (EEA) is the commonest form of endometrial cancer and

associated with exposure to estrogen with high level of estrogen activity. In our study, we would like to

determine the sequence variants and mutations that are associated with estrogen receptor (ER) positive and ER

negative endometrioid endometrial cancer compared to normal endometrium. Twelve fresh frozen endometrial

tissues were obtained (five normal endometrium, four ER positive EEA and three ER negative EEA) from total

hysterectomised patients. Immunohistochemistry was performed on cancer samples to confirm ER status

followed by DNA extraction. Gene variants were characterised using Ion Ampliseq™ Comprehensive Cancer

Panel on Ion Torrent PGM. Nine deleterious mutations were identified in all EEA, which included PDE4DIP

p.R1867C; p.R176C; pK1266E; p.K1222E; p.L83Q, DDR2 p.L420L, MTRR p.I49M; p.I22M, CSF1R

p.H362R; MLL3 p.R284Q, UBR5 p.H757P, TET1 p.I1123M, NUP98 p.A1428A; p.M275I and THBS1

p.Q882H. We identified a novel missense mutation involving E3 ubiquitin protein ligase UBR5 (p.H757;

histidine to proline) that was unique to ER positive sample (75%; n=4) and ER negative sample (67%; n=3).

UBR5 is involved in ubiquitin mediated proteolysis pathway that plays an important role in the regulation of

cell cycle, control of signal transduction pathways, development and differentiation. A novel deleterious

alteration in a tumour suppressor gene DCC (p.G1031R; glycine to arginine) encoding a netrin 1 receptor has

been identified and is unique to ER positive EEA samples (75%; n=4). This protein partially localises to lipid

rafts, and induces apoptosis in the absence of ligand. Deleterious mutation unique to all ER negative EEA

(100%; n=3) have been identified in five genes; FGFR4 p.G388R, EZH2 p.D176H; p.D146H; p.D185H,

TGM7 p.G241E, CDH11 p.M275I, CYP2D6 p.P34S. FGFR4 encodes protein which is a member of fibroblast

growth factor receptor family involved in mitogenesis and differentiation. However, the involvement of these

variants in EEA is yet to be clarified. These preliminary finding prove the utility of targeted next generation

sequencing via the Ampliseq™ Comprehensive Cancer Panel on Ion Torrent platform for rapid mutation

screening of molecular subtypes in EEA. Accurate molecular characterisation between these groups might lead

to improved diagnosis, prognosis prediction and discovery of treatment for endometrial cancer.

P56





Transcript Profiles of Endometrial Cancer Related Gene in Pre- and Post-

Metformin Treatment in Polycystic Ovarian Syndrome

1Ryia Illani Mohd Yunos,

2Nor Azurah Abd Ghani,

2Dahlia Abd Malik,


1Norfilza Mohd Mokhtar

1UKM Medical Molecular Biology Institute, 2Department of Obstetrics & Gynecology, UKM Medical Centre,

The National University of Malaysia (UKM)

ABSTRACT

Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in woman, affecting at least

7-9% of women of reproductive age and accounting for about 75% of anovulatary infertility. PCOS

patients are usually prescribed with Metformin which is used for lowering insulin and blood sugar levels,

helps to regulate menstrual cycles, start ovulation and lower the risk of miscarriage. Clinical studies have

suggested that Metformin may be associated with a decreased risk of developing cancer and with a better

response to chemotherapy. This study was carried out to describe differences in expression of four cancer

related genes in patients under the treatment of Metformin (850 mg) for three months compared with pre-

treatment condition using quantitative PCR (qPCR). These four genes were TP53, CCND2, ACTB and

BCL2. Total RNA from 14 pairs of endometrial tissue section from PCOS patients (pre- and post-

treatment) were extracted and subjected to cDNA synthesis before qPCR. GAPDH is used as a reference

gene in this study. TP53 gene was found to be overexpressed in 8 samples (66.7%) of post-treatment

condition with at least 1.4-fold change (p<0.05) as compared to pre-treatment condition. The remaining

four samples were discovered to be underexpressed (33.3%) of TP53 gene with at least 0.4-fold change

(p<0.05) as compared to pre-treatment condition. ACTB gene was found to be overexpressed in two

samples (16.7%) with at least 0.5-fold change and underexpressed in the remaining ten samples (83.3%)

with at least 0.3-fold change in post-treatment condition as compared to pre-treatment sample (p<0.05).

Five samples (41.7%) showed overexpression of CCND2 with at least 1-fold change and underexpressed

in remaining 58.3% of the sample for at least 0.2-fold change. BCL2 gene was overexpressed in eight

samples (72.7%) with at least 1.4-fold change and underexpressed in five samples (27.3%) with at least

0.2-fold change. Overall, the significant up regulation and down regulation of these genes, may gives an

insight of cancer related gene expression after the treatment of Metformin in PCOS patient and it relations

towards the development of endometrial carcinoma.

http://www.webmd.com/medical_information/health_tools/interactive/ovu_calendar

http://www.webmd.com/infertility-and-reproduction/guide/pregnancy-miscarriage

P57





MicroRNA-31 Inhibits GNA13 Expression and Cancer Cell Invasion in

Breast Cancer

1Suhail Ahmed Kabeer Rasheed,

1Cui Rong Teo, E

1mmanuel Jean Beillard,

1Mathijs Voorhoeve,

1Patrick Casey

1Dept of Cancer and Stem Cell Biology, Duke-NUS graduate Medical School, 8 College Road, 169857 Singapore.

ABSTRACT

Heterotrimeric guanine nucleotide binding proteins (G proteins) transmit a variety of extracellular signals from

cell surface G protein-coupled receptors (GPCRs) to intracellular effector molecules. Heterotrimeric G proteins

are classified according to the α subunit into four subfamilies: Gs, Gi, Gq, and G12. The G12 subfamily, which

is comprised of two members, Gα12 (GNA12) and Gα13 (GNA13), has been implicated in cancer cell

invasion and metastasis. G12 signaling promotes prostate, breast and ovarian cancer cell invasion in vitro, and

these proteins are highly expressed in metastatic cancer tissues. Though it is known that GNA13 is upregulated

in breast cancer tissues, the specific role of GNA13 in breast cancer cells is not well understood. This project

was carried out to determine (1) whether GNA13 expression important for cancer cell invasion and (2) the

mechanism of deregulation of GNA13 expression in breast cancers. Breast cancer cell lines MCF-10a (low

GNA13/low invasiveness) and MDA-MB-231 cell lines (high GNA13/high invasiveness) were used as the

model system. Overexpression of GNA13 in MCF-10a cells induced basal as well as FCS-induced invasion,

whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited basal and FCS-induced invasion.

Since previous studies from our lab using prostate cancer cells showed that microRNAs -182 and -200a

synergistically inhibit the expression of GNA13, we undertook analysis of the potential involvement of

microRNAs in controlling GNA13 expression in these breast cancer cell lines. Expression analysis of miRNAs

predicted to bind the 3’UTR of GNA13 revealed that miR-31 showed an inverse correlation to the protein

expression in MCF-10a and MDA-MB-231 cells. Ectopic expression of miR-31 in MDA-MB-231 cells

significantly reduced GNA13 mRNA and protein levels, as well as GNA13 3’UTR- reporter activity. Further,

expression of miR-31 significantly inhibited MDA-MB-231 cell invasion in an in vitro matrigel invasion

assay, and this effect was partly blocked by ectopic expression of GNA13 in these cells. These data provide

strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms

involving miR-31. Further studies are being performed to identify the role of this miRNA, and those

implicated controlling GNA13 expression in prostate cells, in cancer progression, to determine the importance

of impact on GNA13 expression as an important mediator of this effect.





Identification of Potential Diagnostic Markers in Colorectal Cancer Via

Integrated Epigenomic and Genomic Data

1KS Teow*,

1,2NM Mokhtar,

1NZA Hassan,

1M Ishak,

3I Sagap,

4 IM Rose,

5R Harun,

1R Jamal

UKM Medical Molecular Biology Institute,1 Department of Physiology, Faculty of Medicine, 2 Department of Surgery, Faculty of Medicine,3

Department of Pathology, Faculty of Medicine,4 Department of Medicine, Faculty of Medicine, UniversitiKebangsaan Malaysia,

Kuala Lumpur, Malaysia5

ABSTRACT

Epigenetic alteration is a common phenomenon that contributes to the carcinogenesis in colorectal cancer.

Promoter hypermethylation of tumour suppressor genes leads to transcriptional silencing of the genes.

Integrated data from the epigenome and transcriptome datasets from a similar set of colorectal tissue

samples may explain the complexity of the cancer. We performed methylation profiling using the

Illumina InfiniumHumanMethylation27 BeadChip on 55 paired cancer and adjacent non-cancerous

epithelial cells. Fifteen out of the 55 paired tissues were used for gene expression profiling using the

Affymetrix GeneChip ®

Human Gene 1.0 ST Array. Validation was done on 150 colorectal tissues using

the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique.PCA

and unsupervised hierarchical clustering showed a good separation between cancerous and non-cancerous

samples. Significant genes from both analyses were obtained based on fold-change of at least 2 and FDR

p-value of < 0.05. We identified 1081 differentially hypermethylated CpG sites and 36 hypomethylated

CpG sites. We also found 709 up-regulated and 699 down-regulated genes from the gene expression

profiling. Integrationof these two datasets revealed 32 overlapping genes with 27 being hypermethylated

with down-regulated expression, 4 hypermethylated with up-regulated expression and only one

hypomethylated gene with down-regulated. We identified 95 KEGG pathways with 11 pathways were

related to colorectal cancer. Only one pathway was found to be enriched in the study which was cell

adhesion molecules. We have successfully identified a group of genes that showed both methylation and

gene expression changes in well-representative colorectal cancer tissues. This gives additional insight

regarding the regulation of colorectal cancer-related genes and their underlying mechanisms that

contribute to the colorectal carcinogenesis.

P59





Identification of Potential DNA Methylation Therapeutic Targets in Breast Cancer

via Integrative Genomics Analysis

1*

M Chin, 1,2

NM Mokhtar, 3N Abdullah,

3R Muhammad,

4NA Emran, S

5AM Ali,

6R Harun,

1R Jamal

1UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 2Department of Physiology, Faculty of

Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 3 Department of Surgery, Faculty of Medicine, Universiti Kebangsaan

Malaysia, Kuala Lumpur, Malaysia, 4 Department of Surgery, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia, 5 Department of Pathology,

Faculty of Medicine Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 6Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

ABSTRACT

DNA methylation, an epigenetic event could lead to transcriptional silencing of tumour suppressor genes

and may contribute to breast carcinogenesis. Reversibility of this mechanism can result in gene-re-

expression that lead to normal gene regulation. Therefore, DNA methylation can be served as potential

therapeutic targets in breast cancer. The objectives of this study were to identify the differentially

methylated genes between breast cancer cells and non-cancerous tissues. In addition, we would like to

investigate the regulation of genes as well as molecular pathways that linked these genes in breast cancer

using an integrative approach. DNA methylation profiling was carried out on 76 representative fresh

frozen primary breast tumour tissues and 25 adjacent non-cancerous breast tissues using the Illumina

Infinium® HumanMethylation27 BeadChip. Validation of methylation profiling was performed on 5

genes using Methylation Specific-MLPA. Gene expression profiling was done on 15 breast tumours and 5

adjacent non-cancerous breast tissues using Affymetrix GeneChip® Human Gene 1.0 ST Array. The

overlapped genes between DNA methylation and gene expression datasets were further mapped to KEGG

database to identify the molecular pathways that linked these genes. Supervised hierarchical clustering

analysis revealed 1255 hypermethylation CpG sites and 15 hypomethylation CpG sites. Gene expression

microarray analysis using fold-change of at least 1.5 and p-value with False Discovery Rate < 0.05

identified 404 up-regulated and 463 down-regulated genes. Integration between both datasets identified a

total of 64 genes. Most of the overlapping genes belong to the focal adhesion and extracellular matrix-

receptor interaction that play important roles in breast carcinogenesis. Integrated genomic data from DNA

methylation and gene expression profiling was able to narrow down the genes list with their significant

pathways that contribute to breast cancer. These genes could serve as a potential therapeutic target for

breast cancer.

P60





RNA Interference-Mediated Gene Knockdown on Cell Cycle-related Genes in

Chemosensitized Colorectal Cancer Cell Lines

1*

Tammy Ting Lik Tun; 1,3

Roslan Harun; 1Rahman Jamal and

1,2Norfilza Mohd. Mokhtar;

1UKM Medical Molecular Biology Institute,2Department of Physiology, Faculty of Medicine, 3Department of Medicine, Faculty of Medicine,

Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

ABSTRACT

The development of RNA interference (RNAi) technology has made it possible to suppress the function

of specific molecular targets and we speculate that the use of RNAi together with 5-FU would reduce the

necessary dose of anticancer agents. Integration of publicly available microarray data (Oncomine) with

our current microarray dataset revealed 20 up-regulated genes which involved in cell-cycle function. This

study was performed to investigate their roles following gene silencing on cell viability, cell apoptosis,

cell cycle, cell migration and cell invasion following treatment with 5-FU treatment in human colorectal

cancer cell lines, HCT116 and HT-29. The efficacy of RNAi was assessed by quantitative PCR and

Western blot. Out of 20 genes, RNAi targeting TMEM97, DKC1, ZAK and NME1 showed significant

suppression of cell viability (P < 0.05) compared to the untreated cells on both cell lines. RNAi targeting

DKC1 arrested the HCT116 in the G1 phase of the cell cycle. When combined with 5-FU treatment,

RNAi-mediated DKC1 enhanced chemosensitivity in HCT116 cell by further reduction in cell viability

and arrested cells at S phase of the cell cycle. Significant reduction of HCT116 cell migration was

observed following the RNAi targeting DKC1 and NME1 genes. Silencing of NME1 gene increased the

invasiveness of HT-29 cells relative to the control cells (P < 0.05). However, combination of 5-FU

treatment and RNAi did not give significant result in cells migration and invasion when compared to

RNAi-transfected cells in both HCT116 and HT-29 cells. In conclusion, silencing of DKC1 in

combination with chemotherapy treatment may represent a good strategy to inhibit the growth of

colorectal cancer.

P61





Breast Cancer Mass Localization using Mammogram

Ashwaq Qasem*.SitiNorul Huda Sheikh Abdullah , RizuanaIqbalHussain , NorliaBniti Abdullah , Fuad Ismail,

Tengku siti Meriam Tengku Wook.

Universiti Kebangsaan Malaysia,Malaysia

ABSTRACT

Breast cancer is one of the most dangerous types of the cancer that affect the woman around the world.

Detecting of it in early stage is an important step to reduce the dangerous. . One sign of abnormality that

can appear clearly in mammogram image early is mass. By using mammogram, which is a screening tool

for breast cancer, the abnormalities can be detected up to two years before the specialist. Computer Aid

Detection system (CAD) has been used to speed up the radiologist decision making in detecting

cancerous nodule in human body. CAD is using image processing techniques to analysing mammogram

images and localizing abnormality. In this paper, active contour is used to segment mammogram image

and localize masses and classify it to benign and malignant. Firstly, mammogram image is segmented

using active contour algorithm then features willbe extracted from the segmented images. Finally,

classification of these features into benign and malignant will be done using support vector machine. A

collection of mammogram breast cancer consisting of 40 benign and 40 malignant images has been

obtained from UKM medical center. The experiment, show a significant results in localizing breast

cancer and also classifying cancer and non-cancer mammogram images.

http://www.ftsm.ukm.my/maklumatakademik.php?id=K009682





Gelam Honey Protects Young and Aged Rats from Oxidative Damage and

Modulates the Antioxidant Enzymes Activity

Z Sahhugi1, Z Jubri, NF Rajab

2

1Department of Biochemistry, Faculty of Medicine, National University of Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur,

Malaysia. 2Department of Biomedical Sciences, Faculty of Health Sciences, National University of Malaysia, Jalan Raja Muda Abdul Aziz, 50300

Kuala Lumpur, Malaysia.

ABSTRACT

This study was conducted to evaluate the effect of local gelam honey on oxidative damage of two months

old rats (young group) and 19 months old rats (aged group) and to compare its effect on calorie restriction

(CR) regiment. Thirty-six male Spraque-Dawley rats were divided into young (2 months) and aged (19

months) rats. For each group was further divided into three groups; fed with plain water (control),

supplemented with 2.5g/kg body weight of gelam honey and 40% of CR treatment for 8 months. DNA

damage level was determined by comet assay and plasma malondialdehyde (MDA) by high performance

liquid chromatography (HPLC). The activity of blood antioxidant enzymes were determined by

spectrophotometer. DNA damage was increased in aged group as compared to young group. Gelam honey

supplementation and CR group reduces the DNA damage and plasma MDA level in young and aged

groups. Gelam honey also reduces superoxide dismutase (SOD), catalase (CAT) and glutathione

peroxidise (GPx) activity in young group and increases CAT activity in aged group. Gelam honey reduces

oxidative damage might be via the modulation of antioxidant enzymes activity.

P63





Determining the Cognitive Functions of Different Level of Difficulties Using

Stroop Task : A Preliminary Study

*1Z. M. Mohd Zaki,

1N. F. Abdul Sani,

1W. Z. Wan Ngah,

2M. Mohamad ,

1M. H. Ahmad Damanhuri,

3M. Mazlan.

1Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur,

2Diagnostic Imaging and Radiotherapy Programme, Schools of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti

Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, 3Faculty of Medicine, Universiti Teknologi Mara Sungai Buloh

Campus, Jalan Hospital, 4700 Sungai Buloh, Selangor Darul Ehsan.

ABSTRACT

Aging has become a major concern considering the rapid growth of the older population in the country.

Cases of aging have been commonly connected to major health problem and chronic illnesses, including

cognitive decline. In this preliminary study, the Stroop Task questionnaire was conducted on seven people

with age range from 27 years old to 57 years old. The level of academic is set to a maximum of degree

holder among the respondents. The Stroop Task, which involves the executive function of the brain,

including the inhibitory ability in determining the relevant information and processing speed of an

individual, is divided into five different level of difficulties, in four repetitive sets. The results showed

that there were no significant difference among the different difficulty level of the Stroop Task when

compared between the number of errors with each sets and difficulty levels. However, there were

significance can be observed in the reliability of some of the questionnaires. Statistical method used is a

One Way ANOVA and Chronbach’s alpha with a significant value of p<0.05 and nearest to 1.00

repectively. The insignificant result could happened due to the small number of respondent, which is

N=7. Thus, we suggest to conducting the test on a bigger sample size in the future to observe any

significance of Stroop Task in different age groups.

P64





Gelam Honey Increases Antioxidant Enzymes Activity of Myocardium in Aged

Rats: A Preliminary Study

1SM Hasenan,

1Z Jubri

1Department of Biochemistry, Faculty of Medicine, National University of Malaysia, Jalan Raja Muda Abdul Aziz,

50300 Kuala Lumpur, Malaysia.

ABSTRACT

The study was conducted to determine the effect of local gelam honey on the antioxidant enzymes activity

of myocardium in aged rat. Thirty-two male Spraque-Dawley rats were divided into young (2 months)

and aged (19 months) rats. Each group was further divided into two groups; fed with plain water (control)

and supplemented with 2.5g/kg body weight of gelam honey for 8 months. The activities of superoxide

dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were determined by

spectrophotometer. Antioxidant enzymes activity (SOD, CAT and GPx) of myocardium were increased

significantly (p<0.05) by gelam honey supplementation in young and aged group compared to the control.

Gelam honey increased antioxidant activity by enhancing the enzymatic antioxidant defense. Thus, it

might be effective to protect against age-linked oxidative stress.

P65





Tocotrienol Rich Fraction Post-treatment Improved Proliferative Capacity of

Aging Myoblasts

a*

JJ Lim, aWZWan Ngah,

b,c,dV Mouly,

aNAbdul Karim

aDepartment of Biochemistry, Faculty of Medicine, UniversitiKebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur,

Malaysia, bThérapie des maladies du muscle strié, Institut de Myologie, UM76, Université Pierre et Marie Curie, G.H. Pitié-Salpétrière 47 bd de

l‘hôpital, Paris, France, cINSERM U974, G.H. Pitié-Salpétrière 47 bd de l‘hôpital, Paris, France, and dCNRS UMR 7215, G.H. Pitié-Salpétrière 47 bd de l‘hôpital, Paris, France.

ABSTRACT

Skeletal muscle satellite cells are heavily involved in the regeneration of skeletal muscle in response

tosarcopenia,the aging-related deterioration of skeletal muscle including progressive loss in muscle

strength, mass and regenerative capacity and consequently loss of independency and mobility.Tocotrienol

are renowned for its antioxidant capacity and modulation in cellular aging.In this study, we focusedon the

effect of tocotrienol rich fraction (TRF) towards the regenerative capacity of myoblasts in stress-induced

premature senescence (SIPS). Upon isolation, skeletal muscle satellite cells proliferate in culture as

myoblasts. Myoblasts was grouped as young control, SIPS-induced, TRF control, TRF pre-treatment

(TRF treatment before SIPS-induced) and TRF post-treatment (TRF treatment after SIPS-

induced).Morphological observation, activity of senescence-associated β-galactosidase (SA-β-

galactosidase) and cell proliferation were determined. SIPS-induced myoblasts exhibit senescent-like

morphology, i.e. large flattened cells and prominent intermediate filaments as compared to young spindle-

like myoblasts. The activity of SA-β-galactosidase was significantly increased and the proliferation

capacity was significantly reduced in the SIPS-induced myoblasts as compared to young control.

Interestingly, the activity of SA-β-galactosidase was significantly reduced and cell proliferation was

significantly increased in the post-treatment group as compared to SIPS-induced myoblasts. However,

there was no significant difference in the activity of SA-β-galactosidase and the proliferation capacity of

the pre-treatment group as compared to SIPS-induced myoblasts. We hypothesized that TRF may reverse

the aging of myoblasts through morphological reversal and replenishing the proliferative and regenerative

capacity of the myoblasts. However, further investigation on the mechanism of the action of the TRF in

reversing the aging of myoblasts is needed.

P66





Effects of Tocotrienol-rich Fraction, Alpha-Tocopherol and N-acetylcysteine in

Preventing Replicative Senescence of Human Myoblasts

*SC Khor, WZ Wan Ngah, N Abdul Karim, S Makpol

Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia.

ABSTRACT

Myoblasts are responsible for muscle regeneration in skeletal muscle after injury. In vitro, the

proliferation rate of these cells decreases after certain number of population doublings and finally reaches

replicative senescence. Tocotrienol-rich fraction (TRF), alpha-tocopherol (ATF) and N-acetylcystein

(NAC) have been reported to reduce oxidative stress effectively in vivo or in vitro. This study aimed to

determine the effects of TRF, ATF and NAC in preventing replicative senescence of human myoblasts.

Primary human myoblasts were cultured into young, pre-senescent and senescent. The population

doublings (PD) and myogenic purity of the myoblasts were monitored throughout the study. Cells were

treated with different doses of TRF, ATF and NAC for 24 h. Cells viability and senescence-associated

beta-galactosidase (SA-β-GAL) were determined by using MTS assay and SA-β-GAL staining kit

respectively. Cells morphology was determined by immunocytochemistry method using a specific

antibody for Desmin and observed under a laser scanning confocal microscope. Different doses of TRF,

ATF and NAC were non-cytotoxic to human myoblasts. Cell viability increased significantly with TRF

and ATF treatment at a concentration of 50 ug/ml in young, pre-senescent and senescent myoblasts as

compared to untreated control (p<0.05). NAC treatment increased cell viability in young, pre-senescent

and senescent myoblasts in a dose dependent manner. SA-β-GAL decreased significantly in senescent

myoblasts with TRF, ATF and NAC treatment as compared to untreated control (p<0.05). The typical

replicative senescence morphology was observed in senescent myoblasts. However, treatment with TRF,

ATF and NAC was beneficial in retrieving the young cell morphology in these senescent myoblasts. TRF

and ATF showed the potential in preventing replicative senescence of human myoblasts, which were

comparable to the effects of NAC. These findings may indicate that the anti aging effects of TRF and

ATF in human myoblast could be contributed by their antioxidant properties.

P67

Asia-Pacific Journal of Molecular Medicine 2013




Tocotrienol-Rich Fraction Modulated the Expression of Sirt1, SIRT1 Regulator

and Genes Involved In Cell Cycle Regulation in the Prevention of Cellular

Senescence

*F. Jaafar and S. Makpol

Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz,

53000, Kuala Lumpur, Malaysia.

ABSTRACT

SIRT1 is a key regulator of cellular senescence which can be modulated by natural compounds such as

resveratrol. Tocotrienol-rich fraction (TRF) has been shown to modulate genes and protein expression

thus prevent cellular senescence of human diploid fibroblasts. This study was aimed to determine the

effects of TRF on the expression of genes involved in SIRT1 regulation. SIRT1 gene expression in

primary young human diploid fibroblasts (HDFs) was supressed by siRNA mediated silencing while

SIRT1 activity was inhibited by SIRT1 inhibitor, sirtinol. HDFs were treated with TRF prior or post to

SIRT1 gene suppression or SIRT1 activity inhibition. Young cells which were either transfected with

SIRT1 siRNA or treated with sirtinol exhibited senescence characteristics and positive for SA-β-

galactosidase (SA β-gal) staining. TRF treatment (500 µg/mL) for 24 h however reversed the cell

morphology to young and the percentage of cells positive for SA β-gal was significantly decreased

(p<0.05). At transcriptional level, the expression of SIRT1 gene increased when cells were treated with

TRF after SIRT1 mRNA supression (p<0.05). CDKN2A gene was up regulated (p<0.05) when SIRT1

activity was inhibited and its expression was significantly decreased with TRF treatment (p<0.05). The

expression of E2F1 gene was elevated with post treatment of TRF after SIRT1 mRNA suppression and

SIRT1 activity inhibition. CCND1 was significantly up regulated in senescent HDFs (p<0.05) while

inhibition of SIRT1 expression did not affect the expression of CCND1. Treatment with TRF prior to

SIRT1 mRNA suppression however increased the expression of CCND1 (p<0.05). TRF prevents cellular

senescence of HDFs by modulating SIRT1 and CDKN2A genes and promotes cells proliferation by

inducing E2F1 and CCND1 expression.

P68





Cognitive Performance of Middle Age Rats Using the Morris Water Maze Test:

A Preliminary Report

*1

S.A. Abdul Jalil,1H.S. Hamezah,

1S. Makpol,

1W.Z. Wan Ngah,

1M. Mazlan,

1H. Damanhuri

1Department of Biochemistry, Faculty of Medicine, The National University of Malaysia, Jalan Raja Muda Abdul Aziz

50300 Kuala Lumpur, Malaysia

ABSTRACT

The main aim of the project is to determine the relationship between molecular changes with cognitive

decline. However in this preliminary study only the determination of cognitive decline via Morris water

maze (MWM) has been done. MWM is being used to investigate spatial learning and memory of

laboratory rats. It has been well known that MWM performance may decline with increasing age of the

animals. To measure the cognitive performance of middle age rats, 10 male Sprague Dawley rats (14

months) were tested for open field and water maze test. Exploration of animals was tested during open

field test by placing them in a bare chamber for 5 min. The number of fecal boli, wall supported rearing

and grooming were determined. For water maze task is divided into 4 subtasks such as place navigation (1

– 6 days), probe test (day 7), cued navigation (day 8 & 9) and lastly is working memory version of

MWM. The mean number of fecal boli, wall supported rearing and number of grooming during 5 min

exposure to open field chamber reported 0.5, 12.8 and 4.7 respectively for middle aged rats. Whereas for

place navigation, mean path length and latency were analysed and showed there were significant effected

by day of testing. Number of crossing over target platform and percentage of time in target quadrant

showed no significant difference between probe 1 and 2. Cued navigation showed significant effect day of

mean path length and latency. In working memory, there was no significant effect day of testing in

sample phase was observed and also no significant difference between delay of 30 s and 180 s. Overall,

these data suggest that cognitive performance remain intact after few days of training (place navigation &

probe test), except for working memory as there are not enough groups to be compared.

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